HSV-1 and HSV-2 vaccines and methods of use thereof

ABSTRACT

This invention provides methods of treating, suppressing, inhibiting, reducing an incidence, reducing the pathogenesis of, ameliorating the symptoms of, or ameliorating the secondary symptoms of a primary or recurring Herpes Simplex Virus (HSV) infection, or prolonging the latency to a relapse of an HSV infection, and disorders and symptoms associated with same and inducing an anti-HSV immune response in a subject comprising the step of contacting the subject with a composition comprising a mutant HSV strain comprising an inactivating mutation in a Us8 gene, followed by a second contacting with the composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. patent applicationSer. No. 13/260,835, filed Jan. 20, 2012, which is a National Phaseapplication of PCT International Application Number PCT/US10/29493,International Filing Date Mar. 31, 2010 which claims priority to U.S.patent application Ser. No. 12/415,152, filed on Mar. 31, 2009, now U.S.Pat. No. 8,865,185, which is a continuation-in-part of Ser. No.12/440,223 filed on Jan. 25, 2011, now U.S. Pat. No. 8,871,223, which isa National Phase application of PCT International Application No.PCT/US07/19537, International Filing Date Sep. 7, 2007, which claims thebenefit of U.S. Provisional Patent Application No. 60/842,947 filed Sep.8, 2006 and U.S. Provisional Patent Application No. 60/929,050 filedJun. 11, 2007, which are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention described herein was supported in whole or in part bygrants from the National Institutes of Health (Grant No. R01A133063).The United States government has certain rights in the invention.

FIELD OF INVENTION

This invention provides methods of treating, suppressing, inhibiting,reducing an incidence, reducing the pathogenesis of, ameliorating thesymptoms of, or ameliorating the secondary symptoms of a primary orrecurring Herpes Simplex Virus (HSV) infection, or prolonging thelatency to a relapse of an HSV infection, and disorders and symptomsassociated with same and inducing an anti-HSV immune response in asubject comprising the step of contacting the subject with a compositioncomprising a mutant HSV strain comprising an inactivating mutation in aUs8 gene, followed by a second contacting with the composition

BACKGROUND OF THE INVENTION

Human infection with herpes simplex virus (HSV) type 1 or 2 is typicallyacquired through intimate contact and causes oral and genital lesions.HSV-1 usually causes oral ulcers and HSV-2 normally causes genitalulcers, but the reverse can also occur. A person infected with HSV-1 orHSV-2 will always be a carrier of the virus. After initial infection,lesions heal and HSV exists in a dormant, latent state in sensoryneurons. Periodically, HSV reactivates from latently infected neuronsand causes new ulcers to form at the skin surface. Newborn infants andimmunosuppressed individuals are particularly vulnerable to HSVinfection, often having a disseminated infection with fatal results.Ocular HSV infection, a leading cause of blindness, is another seriousconsequence of the virus. Furthermore, genital HSV infection results ina two-fold increase in HIV transmission rate. Therefore, a vaccine toprevent infection with and transmission of HSV is urgently needed.

SUMMARY OF THE INVENTION

This invention provides methods of treating, suppressing, inhibiting,reducing an incidence, reducing the pathogenesis of, ameliorating thesymptoms of, or ameliorating the secondary symptoms of a primary orrecurring Herpes Simplex Virus (HSV) infection, or prolonging thelatency to a relapse of an HSV infection, and disorders and symptomsassociated with same and inducing an anti-HSV immune response in asubject comprising the step of contacting the subject with a compositioncomprising a mutant HSV strain comprising an inactivating mutation in aUs8 gene, followed by a second contacting with the composition.

In one embodiment, the present invention provides a method of inducingan anti-Herpes Simplex Virus (HSV) immune response in a subjectcomprising the step of contacting a subject with a compositioncomprising a mutant HSV strain, wherein said mutant HSV strain comprisesan inactivating mutation in a Us8 gene, followed by a second contactingof said composition comprising said mutant HSV strain.

In another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject comprisingthe step of administering to said subject a composition comprising amutant HSV strain, wherein said mutant HSV strain comprises aninactivating mutation in a Us8 gene, followed by a second administrationof said composition comprising said mutant HSV strain.

In another embodiment, the present invention provides a method ofsuppressing, inhibiting, or reducing an incidence of a Herpes SimplexVirus (HSV) infection in a subject comprising the step of administeringto said subject a composition comprising a mutant HSV strain, whereinsaid mutant HSV strain comprises an inactivating mutation in a Us8 gene,followed by a second administration of said composition comprising saidmutant HSV strain.

In another embodiment, the present invention provides a method ofreducing the pathogenesis of, ameliorating the symptoms of, amelioratingthe secondary symptoms of, or prolonging the latency to a relapse of aHerpes Simplex Virus (HSV) infection in a subject, comprising the stepof administering to said subject a composition comprising a mutant HSVstrain, wherein said mutant HSV strain comprises an inactivatingmutation in a Us8 gene, followed by a second administration of saidcomposition comprising said mutant HSV strain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. HSV spread in neurons.

FIG. 2. Typical HSV-1 infection of a mouse flank.

FIG. 3. Spectrum of skin disease in the mouse flank/vaccination model.

FIG. 4. Survival of mice following infection with HSV-1(gE null) vaccineor virulent HSV-1(Rescue gE null).

FIG. 5. Primary skin disease after infection with HSV-1(gE null) vaccineor virulent HSV-1(Rescue gE null).

FIG. 6. Secondary (zosteriform) skin disease after infection withHSV-1(gE null) vaccine or virulent HSV-1(Rescue gE null).

FIG. 7. Viral titers in skin after infection with vaccine or virulentHSV-1.

FIG. 8. HSV detection in skin after infection with vaccine or wild-typeHSV-1.

FIG. 9. Viral titers in dorsal root ganglia (DRG) after infection withvaccine or virulent HSV-1.

FIG. 10. Survival of vaccinated or mock-vaccinated mice followingchallenge with 10⁵ pfu of WT HSV-1 (NS).

FIG. 11. Primary skin disease scores in vaccinated mice challenged with10⁵ pfu of WT HSV-1 (NS).

FIG. 12. Viral titers in skin in vaccinated mice challenged with 10⁵ pfuof WT HSV-1 (NS).

FIG. 13. HSV detection in skin of mock-vaccinated or vaccinated mice,challenged with 10⁵ pfu of WT HSV-1 (NS).

FIG. 14. Secondary skin disease in vaccinated mice challenged with 10⁵pfu of WT HSV-1 (NS). N=3.

FIG. 15. Viral titers of ganglia from mock-vaccinated or vaccinatedmice, challenged with 10⁵ pfu of WT HSV-1 (NS). N=3.

FIG. 16. Vaccine protects ganglia from latent infection.

FIG. 17. Cross protection of mice vaccinated with 5×10⁵ pfu HSV-1ΔgEagainst flank challenge with 10⁵ pfu HSV-2(2.12). “Mock Vac” denotesmock vaccination; “DgE Vac” denotes vaccination with 5×10⁵ pfu HSV-1ΔgE.Error bars represent the Standard Error of the Mean (“SEM”).

FIG. 18. Protection of mice vaccinated with 5×10⁵ pfu HSV-1ΔgE againstlatency following flank challenge with 5×10⁵ pfu HSV-1(KOS). Error barsrepresent the SEM.

FIG. 19A and FIG. 19B. Protection of mice vaccinated with 5×10⁵ pfuHSV-1ΔgE against death, visible Disease and Extensive Viral ReplicationFollowing Vaginal Challenge with 10⁵ pfu HSV-1(NS). FIG. 19A. Toppanel-survival curves; bottom panel-viral titer as assessed by vaginalswabs. FIG. 19B. Photographs of mice on day 8 post-challenge. Errorbars: SEM.

FIG. 20. Protection of mice vaccinated with 5×10⁵ pfu HSV-1ΔgE bydifferent routes (“Ep. Scar.”: epidermal scarification; “SubQ”:subcutaneously; IM: intramuscular) against flank challenge with 10⁵ pfuHSV-1(NS). “DgE Vac” denotes HSV-1ΔgE; Error bars represent the SEM.

FIG. 21. Induction of neutralizing antibody response in mice vaccinatedwith 5×10⁵ pfu HSV-1ΔgE by different routes. Percentages depicted arecompared to serum from mock-vaccinated mice. n=3 (Ep. Scar & Sub Q), n=2(IM), assays were done in duplicate. Error bars represent the SEM.

FIG. 22. Protection of mice vaccinated with 5×10⁵ pfu HSV-1ΔgE againstflank challenge with 10⁵ pfu HSV-1(NS, F or 17). Error bars representthe SEM.

FIG. 23. Protection of mice vaccinated with 5×10⁵ pfu HSV-1ΔgE againstflank challenge with 10⁵-7 pfu HSV-1(NS). Error bars represent the SEM.

FIG. 24A. Western blot to detect gD (Us6) and gI (Us7) in infected cellextracts. FIG. 24B. Stability of the KOS-gDA3C virus in vitro. An Ssp1digest of a PCR-amplified gD gene fragment of KOS or KOS-gDA3C. FIG.24C. Stability of the KOS-gDA3C mutant virus in vivo. A PCR-amplified gDfragment obtained from the DRG of KOS-gDA3C-infected mice were cut withSsp1 or left uncut.

FIG. 25. Entry of KOS, rKOS-gDA3C and KOS-gDA3C virus into (FIG. 25A)Vero, (FIG. 25B) B78-H1, (FIG. 25C) A10 or (FIG. 25D) C10 cells. Resultsare the mean+SE of three separate infections each done in triplicate.

FIG. 26. Single-step (FIG. 26A, FIG. 26B) and multi-step (FIG. 26C, FIG.26D) growth curves of KOS, rKOS-gDA3C and KOS-gDA3C performed in A10(FIG. 26A, FIG. 26C) or C10 (FIG. 26B, FIG. 26D) cells. Results are themean+SE of three separate infections.

FIG. 27. Disease in the murine flank model. Inoculation (FIG. 27A) andzosteriform (FIG. 27B) site disease scores in mice inoculated with 5×10⁵PFU of KOS, rKOS-gDA3C or KOS-gDA3C. Error bars represent SE. FIG. 27C.Photographs of mice flanks taken 10 days post-infection with KOS,rKOS-gDA3C, or KOS-gDA3C.

FIG. 28. Virus titers and genome copy numbers in DRG. DRG were dissectedfrom mice infected with KOS, rKOS-gDA3C or KOS-gDA3C and assayed forvirus titers (FIG. 28A) or viral genome copy number (FIG. 28B). Resultsrepresent the mean+SE.

FIG. 29. Prior infection with KOS-gDA3C protects against WT HSV-1challenge. Results represent mean disease scores+SE at the inoculation(FIG. 29A) and zosteriform (FIG. 29B) sites from days 3-7post-infection. DRG viral titers (FIG. 29C) and genome copy number (FIG.29D) were measured 5 days post-challenge with NS. Results represent themean+SE.

FIG. 30. Model for KOS-gDA3C infection in mice. KOS infects epithelialcells (E) and produces disease at the inoculation site. The virusspreads to neurons (N) in the DRG, replicates and spreads to adjacentneurons and then travels back to epithelial cells in the skin to causezosteriform disease. KOS-gDA3C is impaired in entry and infects fewerepithelial cells, which results in fewer neurons becoming infected inthe DRG. The defect in entry also reduces infection of adjacent neuronsin the DRG and results in reduced zosteriform disease.

FIG. 31A. Alignment of HSV-1(NS) gE (SEQ ID NO: 2) with HSV-2(HG52) gE(SEQ ID NO: 18). FIG. 31B. Alignment of HSV-2(2.12) gE (SEQ ID NO: 6)with HSV-2 (HG52) gE (SEQ ID NO: 18). FIG. 31C. Strategy for generationof gE-2 deletion.

FIG. 32. A schematic diagram of HSV-2ΔgE(gfp) deletion and insertion ofgfp2 cassette under the control of a CMV promoter. The gfp2 cassette,which allows for screening of recombinant viruses by fluorescence, isinserted in the US8 reading frame just after the bases encoding aminoacid 123. The portion of the US8 gene encoding the 156 c-terminal aminoacids of gE remain but are not expressed. The flanking regions used forthe recombination are indicated. The mutation was made in wild-typestrain HSV-2(2.12).

FIG. 33. Characterization of HSV-2ΔgE(gfp) protein expression. Westernblot of protein isolated from Vero cells infected with HSV-2(2.12)(WT)or HSV-2ΔgE(gfp) mutants and stained with HSV-2 antibodies raisedagainst VP5, gE, US9 and gD.

FIG. 34. Growth of HSV-2ΔgE(gfp) in vitro. In vitro single-step growthkinetics of HSV-2ΔgE(gfp) and WT HSV-2 in vitro in both epithelial (Verocells) (FIG. 34A) and primary neuronal (superior cervical ganglia fromrat embryos) (FIG. 34B) cell lines. For growth curves, cells wereinfected at an MOI of 3.0 and the zero hour time-point collectedimmediately. After a 1 hour incubation, cells were acid washed with a pH3.0 citrate buffer and the 1 hour time-point was collected. Samplescollected were titered on Vero cells by plaque assay. (FIG. 34C) Plaquesizes in Vero cells were measured at 96 hpi. 25 plaques were averagedfor each virus.

Statistics Table Groups FIG. P value different Statistical Test 34A—Verosingle- =0.2938 No 2-way ANOVA step growth 34B—SCG single =0.8907 Not-test step growth 34C—Plaque size <0.0001 Yes 2-way ANOVA

FIG. 35. Mouse retinal infection with HSV-2ΔgE(gfp). Thin cryosectionswere made of retina (FIG. 35A) and optic nerve (FIG. 35B) followingmouse retina infection with 4×10⁵ PFU of either HSV-2(2.12) orHSV-2ΔgE(gfp). Representative immunofluoresence images from three miceper data point on days 3 and 5 are shown and use an anti-HSV-2polyclonal (DAKO) and a Dapi nuclear stain.

FIG. 36. Anterograde and retrograde retinorecipient areas of the brainfollowing HSV-2ΔgE(gfp) retina infection of the mouse. Mouse retinaswere infected with 4×10⁵ PFU HSV-2ΔgE(gfp) or HSV-2(2.12). Brains werecryosectioned and stained with anti-HSV-2 rabbit polyclonal primaryantibody and goat anti-rabbit HRP conjugated secondary antibody.Representative sections are shown from infection of 3 mice per virusstrain.

FIG. 37. Safety of HSV-2ΔgE(gfp) in the mouse flank model. One day priorto infection, hair was removed from the right flank of mice usingclippers and depilatory cream (Nair™). The following day, mice wereinfected by scarification on denuded flank skin with 5×10⁵ pfuHSV-2(2.12) or HSV-2ΔgE(gfp) by making 60 gentle scratches in severaldifferent directions on a 1 cm square area of the skin. Mice weremonitored daily for (A) survival, (B) inoculation site disease and (C)zosteriform disease. Disease was scored on a scale of 0 (no disease) to4 (most severe). There were 5 mice per group. (D) A representative photofrom each group taken on day 7 post-inoculation is shown. The boxed areaof each picture is the site of inoculation and the arrows indicatezosteriform disease. Statistics Table—2-Way ANOVA (comparisons withp<0.05 are considered significantly different)

FIG. 2.12 vs. ΔgE ΔgE vs. Mock 37B—inoculation P < 0.0001 p = 0.0046site disease 37C—zosteriform p < 0.0001 N/A disease

FIG. 38. Virus yield of HSV-2ΔgE(gfp) in skin and dorsal root ganglion(DRG) following mouse flank scarification. One day prior to infection,hair was removed from the right flanks of mice using clippers anddepilated with depilatory cream (Nair™). The following day, mice wereinfected by scarification on denuded flank skin with 5×10⁵ pfuHSV-2(2.12) or HSV-2ΔgE(gfp) by making 60 gentle scratches in severaldifferent directions on a 1 cm square area of the skin. Mice weresacrificed at intervals to evaluate viral titers in the skin at the siteof inoculation. Skin at the site of inoculation (A) and DRG (B) wereremoved from groups of mice (n=3) on days 1, 3, 6 and 8. Tissues werepulverized and the viral content was quantified by plaque assay.p<0.0001 for skin titers and p=0.0006 for DRG titers. There were 3 micefor each data point. The limit of detection for the titering assay was 5pfu.

FIG. 39. Safety and virus yield of HSV-1 and HSV-2 WT and vaccinestrains in the mouse vaginal model. HSV. Pathogenicity (A), safety (B),and replication (C) of HSV-1 and HSV-2 wild-type and vaccine strainswere assessed in the mouse vaginal model. Mice were treated with DepoProvera and infected 5 days later with 5×10⁵ pfu of each virus. Micewere monitored daily for survival and scored for disease. The limit ofdetection for the titering assay was 20 pfu. There were 5 mice pergroup. Statistics Table—2-Way ANOVA (comparisons where p<0.05 areconsidered significantly different).

FIG. NS vs. gEnull 2.12 vs. ΔgE-2 39B—disease scores P < 0.0001 P <0.0001 39C—swab titers p = 0.0053 p = 0.0004

FIG. 40. Intramuscular (IM) and subcutaneous (subQ) HSV-2ΔgE(gfp)vaccine efficacy following challenge with HSV-2(MS) in the mouse flankmodel; protection against death and disease. Mice were vaccinated with5×10⁵ pfu HSV-2ΔgE(gfp) or mock vaccinated either intra-muscularly (IM)in the right rear thigh or subcutaneously (SubQ) in the neck scruff. Oneday prior to challenge, hair was removed from the right flanks of miceusing clippers and depilated with depilatory cream (Nair™). Thefollowing day, 28 days after vaccination, mice were challenged by flankscarification on denuded flank skin with 5×10⁵ pfu (1,736 LD50s)HSV-2(MS) by making 60 gentle scratches in several different directionson a 1 cm square area of the skin. Mice were monitored daily forsurvival (A), inoculation site disease (B) and zosteriform disease (C).Disease was scored on a scale of 0 (no disease) to 4 (most severe).There were 5 mice per group. Statistics Table—2-Way ANOVA (comparisonswhere p<0.05 are considered significantly different).

IM vs. IM vs. SubQ vs. IM vs. SubQ vs FIG. SubQ mock vac mock vac mockchall mock chall 40B— p = 0.8732 p = 0.0335 P < 0.0001 p = 0.9925 p =0.0789 inoculation site disease 40C— p = 0.9999 p = 0.0012 p = 0.0112 p= 0.99993 p = 0.9456 zosteriform disease

FIG. 41. HSV-2ΔgE(gfp) vaccine efficacy (IM) following HSV-2(MS)challenge in the mouse flank model; protection of tissues. Mice werevaccinated IM with 5×10⁵ pfu HSV-2ΔgE(gfp) or mock vaccinated. 28 dayslater, mice were challenged by flank scarification on denuded flank skinwith 5×10⁵ pfu (1,736 LD50s) HSV-2(MS). Skin at the site of inoculationand DRG were removed from groups of mice (n=3) on days 1, 3, 6 and 8.Tissues were pulverized and the viral content was quantified by plaqueassay. Mice were sacrificed at intervals to evaluate viral titers in theskin at the site of inoculation (A) and the DRG (B). There were 3 micefor each data point. The limit of detection for the titering assay was 5pfu. Statistics Table—2-Way ANOVA (comparisons where p<0.05 areconsidered significantly different).

FIG. p= 41A—skin titers P < 0.0001 41B—DRG titers p = 0.0011

FIG. 42. Vaccine efficacy (SubQ/IM) following challenge with 50 LD50s ofHSV-2(MS) in the mouse vaginal model. Mice were vaccinated IM or SubQwith 5×10⁵ pfu HSV-2ΔgE(gfp) or mock vaccinated. Five days prior tochallenge, mice were treated with Depo Provera. Twenty-eight daysfollowing vaccination, mice were challenged by vaginal instillation of250 pfu (50 LD50s) HSV-2(MS). Mice were monitored daily for disease andsurvival. Disease was scored on a scale of 0 (no disease) to 4 (mostsevere disease). Vaginal swab samples were collected daily and assayedby plaque assay to quantify virus. Mice were monitored daily forsurvival (A) and scored for disease (B). Mice were swabbedintra-vaginally on days 1-7 (C). The limit of detection for the titeringassay was 20 pfu. There were 5 mice per group. Statistics Table—2-WayANOVA (comparisons where p<0.05 are considered significantly different).

FIG. IM vs. SubQ IM vs. mock SubQ vs. mock 42B—Disease Scores p = 0.0099P < 0.0001 P < 0.0001 42C—Swab Titers p = 0.0005 P < 0.0001 P < 0.0001

FIG. 43. Protection against vaginal challenge with a 10⁴ LD50 dose ofHSV-2(MS) after one or two doses of HSV-2ΔgE(gfp). Mice were vaccinatedIM in the right hind leg gastrocnemius muscle with either one or twodoses (three weeks apart) of 5×10⁵ pfu HSV-2ΔgE(gfp) or mock vaccinated.Five days prior to challenge, mice were treated with Depo Provera.Twenty-eight days following the date of the second vaccination, micewere challenged by vaginal instillation of 5×10⁴ pfu (10⁴ LD50s)HSV-2(MS) Mice were monitored daily for survival (A) and scored fordisease (B). Disease was scored on a scale of 0 (no disease) to 4 (mostsevere disease). Vaginal swab samples were collected daily and assayedby plaque assay to quantify virus (C). n=5 per group. The limit ofdetection for the titering assay was 20 pfu. There were 5 mice pergroup. (D) Photos from each mouse taken on day 7 post-inoculation areshown. Scores given for each mouse on day 7 are indicated below eachphoto. Statistics Table—2-Way ANOVA (comparisons where p<0.05 areconsidered significantly different).

FIG. 1X vs. 2X 1X vs. mock 2X vs. mock 43B—Disease Scores p = 0.1024 P <0.0001 P < 0.0001 43C—Swab Titers P < 0.0001 P < 0.0001 P < 0.0001

FIG. 44. Shows safety evaluation of HSV-2ΔgE(gfp) in BALB/c and SCIDmice following intramuscular (IM), intravenous (IV), intracranial, andintravaginal inoculation.

FIG. 45. Shows antibody response to HSV-2 gD measured by ELISA after oneor two immunizations with HSV-2ΔgE(gfp).

FIG. 46. Shows antibody response to HSV-2 gC measured by ELISA after oneor two immunizations with HSV-2ΔgE(gfp).

FIG. 47. Shows neutralizing antibody response after one or twoimmunizations with HSV-2ΔgE(gfp).

FIG. 48. Shows HSV-2ΔgE(gfp) administered as a prophylactic vaccine atvarying immunizing doses in female BALB/c mice. A. shows survival ofanimals following administration of varying doses of the gE2-nullstrains and mock control. B. Animals were scored for vaginal disease ona scale of 0-4, where 0 is no disease, and one point was assigned foreach of the following: erythma/swelling, exudate, hair loss in theperineal area, and ulcers or necrosis in the vaginal area. C. Animalswere evaluated for vaginal titers. D. Animals were evaluated for viraltiters or viral DNA in dorsal root ganglia (DRG) 4 days post-infectionor 35 days post-infection (labeled as latent viral load).

FIG. 49. Assessment of DRG at day 35 for wild-type or vaccine strainDNA.

FIG. 50. Evaluation of HSV-2ΔgE(gfp) as a prophylactic vaccine inHartley Strain guinea pigs. A. mock immunized guinea pigs challengedwith 5×10³ or 5×10⁵ PFU. B. Vaginal disease scores in mock immunizedanimals and in animals immunized with HSV-2ΔgE(gfp). C. Vaginal titersin mock immunized animals and in animals immunized with HSV-2ΔgE(gfp).D. Shows the number of recurrences and the number of animals having arecurrence between days 15-49 post-infection. E. real-time qPCR forHSV-2 DNA.

FIG. 51. Immunization with HSV-2ΔgE(gfp) as a therapeutic vaccine totreat recurrent infections in guinea pigs. A. ELISA of anti-gC-2antibodies. B. ELISA of anti-gD-2 antibodies. C. guinea pigs immunizedwith HSV-2ΔgE(gfp) or mock immunized. D. real-time qPCR for HSV-2 DNA.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods of vaccinating a subject against HerpesSimplex Virus (HSV) infection and disorders and symptoms associated withsame, and impeding, inhibiting, reducing the incidence of, andsuppressing HSV infection, neuronal viral spread, formation ofzosteriform lesions, herpetic ocular disease, herpes-mediatedencephalitis, and genital ulcer disease in a subject, comprising thestep of contacting the subject with a mutant strain of the HSV,containing an inactivating mutation in a gene encoding a gE, gI, Us9,other protein, or combinations thereof. In another embodiment, themutant strain of the HSV, comprises an inactivating mutation in a geneencoding gE, which in one embodiment, is a gE null mutation. In anotherembodiment, the present invention provides pharmaceutical compositionscomprising a mutant strain of HSV which comprises an inactivatingmutation in a gene encoding gE, which in one embodiment, is a gE nullmutation. In another embodiment, this invention provides pharmaceuticalcompositions comprising a mutant strain of HSV which comprises aninactivating mutation in a gene encoding a gE, gI, Us9, other protein,or combinations thereof.

In one embodiment, the present invention provides a method of inducingan anti-Herpes Simplex Virus (HSV) immune response in a subjectcomprising the step of contacting a subject with a compositioncomprising a mutant HSV strain, wherein said mutant HSV strain comprisesan inactivating mutation in a Us8 gene, followed by a secondadministration of said composition comprising said mutant HSV strain.

In another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject comprisingthe step of administering to said subject a composition comprising amutant HSV strain, wherein said mutant HSV strain comprises aninactivating mutation in a Us8 gene, followed by a second administrationof said composition comprising said mutant HSV strain.

In another embodiment, the present invention provides a method ofsuppressing, inhibiting, or reducing an incidence of a Herpes SimplexVirus (HSV) infection in a subject comprising the step of administeringto said subject a composition comprising a mutant HSV strain, whereinsaid mutant HSV strain comprises an inactivating mutation in a Us8 gene,followed by a second administration of said composition comprising saidmutant HSV strain.

In one embodiment, the present invention provides a method of reducingthe pathogenesis of, ameliorating the symptoms of, ameliorating thesecondary symptoms of, or prolonging the latency to a relapse of aHerpes Simplex Virus (HSV) infection in a subject, comprising the stepof administering to said subject a composition comprising a mutant HSVstrain, wherein said mutant HSV strain comprises an inactivatingmutation in a Us8 gene, followed by a second administration of saidcomposition comprising said mutant HSV strain.

In another embodiment, the present invention provides a method ofinducing an anti-Herpes Simplex Virus (HSV) immune response in a subjectcomprising the step of contacting a subject with a compositioncomprising a mutant HSV strain, wherein said mutant HSV strain comprisesan inactivating mutation in a Us8 gene and wherein said composition isadministered in a first inoculation or “priming inoculation” and in asecond inoculation or “boosting inoculation”.

In another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject comprisingthe step of administering to said subject a composition comprising amutant HSV strain, wherein said mutant HSV strain comprises aninactivating mutation in a Us8 gene and wherein said composition isadministered in a first inoculation or “priming inoculation” and in asecond inoculation or “boosting inoculation”.

In another embodiment, the present invention provides a method ofsuppressing, inhibiting, or reducing an incidence of a Herpes SimplexVirus (HSV) infection in a subject comprising the step of administeringto said subject a composition comprising a mutant HSV strain, whereinsaid mutant HSV strain comprises an inactivating mutation in a Us8 geneand wherein said composition is administered in a first inoculation or“priming inoculation” and in a second inoculation or “boostinginoculation”.

In another embodiment, the present invention provides a method ofreducing the pathogenesis of, ameliorating the symptoms of, amelioratingthe secondary symptoms of, or prolonging the latency to a relapse of aHerpes Simplex Virus (HSV) infection in a subject, comprising the stepof administering to said subject a composition comprising a mutant HSVstrain, wherein said mutant HSV strain comprises an inactivatingmutation in a Us8 gene and wherein said composition is administered in afirst inoculation or “priming inoculation” and in a second inoculationor “boosting inoculation”.

In one embodiment, the present invention provides a method ofvaccinating a subject against an HSV infection, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein.

In another embodiment, the present invention provides a method ofimpeding HSV-1 infection in a subject, comprising the step of contactingthe subject with a mutant HSV strain, wherein the mutant strain containsan inactivating mutation in a Us8 gene encoding a gE protein.

In another embodiment, the present invention provides a method ofimpeding HSV-2 infection in a subject, comprising the step of contactingthe subject with a mutant HSV strain, wherein the mutant strain containsan inactivating mutation in a Us8 gene encoding a gE protein.

“HSV-1” refers, in one embodiment, to a Herpes Simplex Virus 1. Inanother embodiment, the term refers to a KOS strain. In anotherembodiment, the term refers to an F strain. In another embodiment, theterm refers to an NS strain. In another embodiment, the term refers to aCL101 strain. In another embodiment, the term refers to a “17” strain.In another embodiment, the term refers to a “17+syn” strain. In anotherembodiment, the term refers to a MacIntyre strain. In anotherembodiment, the term refers to an MP strain. In another embodiment, theterm refers to an HF strain. In another embodiment, the term refers toany other HSV-1 strain known in the art.

“HSV-2” refers, in one embodiment to a Herpes Simplex Virus 2. Inanother embodiment, the term refers to an HSV-2 333 strain. In anotherembodiment, the term refers to a 2.12 strain. In another embodiment, theterm refers to an HG52 strain. In another embodiment, the term refers toan MS strain. In another embodiment, the term refers to an 186 strain.In another embodiment, the term refers to a G strain. In anotherembodiment, the term refers to any other HSV-2 strain known in the art.

In another embodiment, the present invention provides a method ofimpeding primary HSV infection in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the present invention provides a methodof impeding primary HSV-1 infection in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein In another embodiment, the present invention provides a methodof impeding primary HSV-2 infection in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein

The terms “impeding HSV infection” and “impeding primary HSV infection”refer, in one embodiment, to decreasing the titer of infectious virus by90%. In another embodiment, the titer is decreased by 50%. In anotherembodiment, the titer is decreased by 55%. In another embodiment, thetiter is decreased by 60%. In another embodiment, the titer is decreasedby 65%. In another embodiment, the titer is decreased by 70%. In anotherembodiment, the titer is decreased by 75%. In another embodiment, thetiter is decreased by 80%. In another embodiment, the titer is decreasedby 85%. In another embodiment, the titer is decreased by 92%. In anotherembodiment, the titer is decreased by 95%. In another embodiment, thetiter is decreased by 96%. In another embodiment, the titer is decreasedby 97%. In another embodiment, the titer is decreased by 98%. In anotherembodiment, the titer is decreased by 99%. In another embodiment, thetiter is decreased by over 99%.

In another embodiment, the terms refer to decreasing the extent of viralreplication by 90%. In another embodiment, replication is reduced by50%. In another embodiment, replication is reduced by 55%. In anotherembodiment, replication is reduced by 60%. In another embodiment,replication is reduced by 65%. In another embodiment, replication isreduced by 70%. In another embodiment, replication is reduced by 75%. Inanother embodiment, replication is reduced by 80%. In anotherembodiment, replication is reduced by 85%. In another embodiment,replication is reduced by 92%. In another embodiment, replication isreduced by 95%. In another embodiment, replication is reduced by 96%. Inanother embodiment, replication is reduced by 97%. In anotherembodiment, replication is reduced by 98%. In another embodiment,replication is reduced by 99%. In another embodiment, replication isreduced by over 99%.

Methods for measuring HSV infection are well known in the art, andinclude, in one embodiment, determination of appearance and severity ofskin lesions and viral-mediated illness (Examples 1 and 4). Otherembodiments of methods for measuring viral infection are described, forexample, in Burgos J S et al. (Herpes simplex virus type 1 infection viathe bloodstream with apolipoprotein E dependence in the gonads isinfluenced by gender. J Virol. 2005 February; 79(3):1605-12) and Parr MB et al. (Intravaginal administration of herpes simplex virus type 2 tomice leads to infection of several neural and extraneural sites. JNeurovirol. 2003 December; 9(6):594-602). Other methods of determiningthe extent of HSV replication and HSV infection are well are described,for example, in Lambiase A et al. (Topical treatment with nerve growthfactor in an animal model of herpetic keratitis. Graefes Arch Clin ExpOphthalmol. 2007 May 4), Ramaswamy M et al. (Interactions and managementissues in HSV and HIV coinfection. Expert Rev Anti Infect Ther. 2007April; 5(2):231-43), and Jiang C et al. (Mutations that decrease DNAbinding of the processivity factor of the herpes simplex virus DNApolymerase reduce viral yield, alter the kinetics of viral DNAreplication, and decrease the fidelity of DNA replication. J Virol. 2007April; 81(7):3495-502).

In one embodiment, vaccination with gE-null HSV strains of the presentinvention protects against subsequent infection with virulent HSV. Inanother embodiment, the vaccination protects against disease caused byvirulent HSV. In another embodiment, the vaccine strain does not itselfcause significant disease, which in one embodiment is herpes (Examples1, 4, and 19), and, in another embodiment, the vaccine strain does notitself result in significant symptoms.

“Inactivating mutation” in gE refers, in one embodiment, to a mutationthat abrogates HSV neuronal spread. In another embodiment, the termsrefer to a mutation that abrogates cell-to-cell spread of HSV. Inanother embodiment, the terms refer to abrogation of spread along axons.In another embodiment, the spread is retrograde (defined herein below).In another embodiment, the spread is anterograde (defined herein below).In another embodiment, spread in both anterograde and retrogradedirections is abrogated.

“Anterograde” refers, in one embodiment, to spread from ganglia to skin.In another embodiment, the term refers to spread from the cell bodytowards the axon. In another embodiment, the term refers to any otherdefinition accepted in the art.

“Retrograde” refers, in one embodiment, to spread from the site ofinfection to ganglia. In another embodiment, the term refers to spreadfrom the axon towards the cell body. In another embodiment, the termrefers to any other definition accepted in the art.

In one embodiment, a “defect” or “deficiency” describes an impairment,which in one embodiment, refers to a 10%, 25%, 40%, 50%, 60%, 75%, or90% decrease in a particular function.

In one embodiment, neuronal spread is decreased by 90%. In anotherembodiment, neuronal spread is decreased by 60%. In another embodiment,the reduction is 65%. In another embodiment, the reduction is 70%. Inanother embodiment, the reduction is 75%. In another embodiment, thereduction is 80%. In another embodiment, the reduction is 85%. Inanother embodiment, the reduction is 95%. In another embodiment, thereduction is 96%. In another embodiment, the reduction is 97%. Inanother embodiment, the reduction is 98%. In another embodiment, thereduction is 99%. In another embodiment, the reduction is over 99%.

In one embodiment, the term refers to abrogating the ability of gEprotein to sequester host anti-HSV antibodies. In another embodiment,sequestration of anti-HSV antibodies by gE is reduced by 90%. In anotherembodiment, sequestration is reduced by 50%. In another embodiment, thereduction is 65%. In another embodiment, the reduction is 70%. Inanother embodiment, the reduction is 75%. In another embodiment, thereduction is 80%. In another embodiment, the reduction is 85%. Inanother embodiment, the reduction is 95%. In another embodiment, thereduction is 96%. In another embodiment, the reduction is 97%. Inanother embodiment, the reduction is 98%. In another embodiment, thereduction is 99%. In another embodiment, the reduction is over 99%.

In one embodiment, the term refers to abrogating the ability of gEprotein to bind IgG monomers. In another embodiment, binding of IgGmonomers by gE is reduced by 90%. In another embodiment, binding isreduced by 50%. In another embodiment, the reduction is 65%. In anotherembodiment, the reduction is 70%. In another embodiment, the reductionis 75%. In another embodiment, the reduction is 80%. In anotherembodiment, the reduction is 85%. In another embodiment, the reductionis 95%. In another embodiment, the reduction is 96%. In anotherembodiment, the reduction is 97%. In another embodiment, the reductionis 98%. In another embodiment, the reduction is 99%. In anotherembodiment, the reduction is over 99%.

In one embodiment, the term refers to abrogating the ability of gEprotein to bind IgG complexes. In another embodiment, binding of IgGcomplexes by gE is reduced by 90%. In another embodiment, binding isreduced by 50%. In another embodiment, the reduction is 65%. In anotherembodiment, the reduction is 70%. In another embodiment, the reductionis 75%. In another embodiment, the reduction is 80%. In anotherembodiment, the reduction is 85%. In another embodiment, the reductionis 95%. In another embodiment, the reduction is 96%. In anotherembodiment, the reduction is 97%. In another embodiment, the reductionis 98%. In another embodiment, the reduction is 99%. In anotherembodiment, the reduction is over 99%.

In one embodiment, an inactivating mutation in gE comprises a deletionof amino acids 124-508. In another embodiment, an inactivating mutationin gE comprises a deletion of amino acids 110-500. In anotherembodiment, an inactivating mutation in gE comprises a deletion of aminoacids 1-552. In another embodiment, an inactivating mutation in gEcomprises a deletion of amino acids 1-50. In another embodiment, aninactivating mutation in gE comprises a deletion of amino acids 1-100.In another embodiment, an inactivating mutation in gE comprises adeletion of amino acids 1-250. In another embodiment, an inactivatingmutation in gE comprises a deletion of amino acids 100-300. In anotherembodiment, an inactivating mutation in gE comprises a deletion of aminoacids 1-400. In another embodiment, an inactivating mutation in gEcomprises a deletion of amino acids 200-500. In another embodiment, aninactivating mutation in gE comprises a deletion of amino acids 24-71.In another embodiment, an inactivating mutation in gE comprises adeletion of amino acids 30-508. In another embodiment, an inactivatingmutation in gE comprises a deletion of approximately amino acids 40-70.In another embodiment, an inactivating mutation in gE comprisesinsertion of a non-native sequence into a portion of the gene encodinggE, wherein said gE is inactivated as a result. In another embodiment,an inactivating mutation in gE comprises substitution of amino acidresidues, such as a substitution of polar for non-polar residues,non-polar for polar residues, charged for uncharged residues, positivelycharged for negatively charged residues, or vice versa, or a combinationthereof, as is known to one of skill in the art. In another embodiment,an inactivating mutation in gE consists essentially of any of themutations listed hereinabove. In another embodiment, an inactivatingmutation in gE consists of any of the mutations listed hereinabove. Inanother embodiment, an inactivating mutation in gE or other protein asdescribed in the instant invention for a first HSV strain may also bemutated in an equivalent location of the corresponding protein in asecond HSV strain, wherein the equivalent location of the insertion,deletion or substitution may be inferred by sequence alignment, as iswell known in the art, wherein the region that aligns with the sequenceof the mutation in the first strain would be mutated in the secondstrain.

“Inactivating mutation” in gD refers, in one embodiment, to a mutationthat inhibits protein/receptor interactions, which in one embodiment isan interaction with a HVEM cell receptor, a nectin-1 cell receptor, orboth, and in another embodiment, to a mutation that inhibits viral entryinto a cell, inhibits downstream activation of gB, gH, and gL, inhibitsfusion of the viral envelope with cell membrane, or a combinationthereof. In one embodiment, an inactivating mutation in gD is in theamino terminus of the gD peptide, which in one embodiment is residue1-15, and in another embodiment, the mutation inhibits formation of ahairpin loop structure when gD is bound to HVEM. In another embodiment,the mutation is at amino acids 3, 38, or both, and in one embodiment,alanine and tyrosine residues at those locations are replaced withcysteine residues (A3C/Y38C) to create a 3-38 disulfide bond and/or afixed hairpin loop at the amino terminus.

In one embodiment, inactivating mutations of the present invention areaccomplished using tools known in the art. In one embodiment, thenucleic acids used in this invention and those encoding proteins of andfor use in the methods of the present invention can be produced by anysynthetic or recombinant process such as is well known in the art.Nucleic acids can further be modified to alter biophysical or biologicalproperties by means of techniques known in the art. For example, thenucleic acid can be modified to increase its stability against nucleases(e.g., “end-capping”), or to modify its lipophilicity, solubility, orbinding affinity to complementary sequences. In another embodiment,transposons may be used to create inactivating mutations of a gene,where in one embodiment, the transposon may be Tn551, Minos, Hermes orpiggyback. In another embodiment, the transposon may be AT-2 (tyl basedtransposon, Perkin Elmer; Devine et al. (1997) Genome Res. 7:551-563),GPS-1 (New England Biolabs), GPS-2 (New England Biolabs), EZ::tn (Tn5based transposon, Epicenter Technologies), SIF (Tn7 based transposon,Biery et al. (2000) Nucl Acid Res 28:1067-1077), or Mu (Finnzymes, Haapaet al. (1999) Nucl Acid Res 13:2777-2784). In one embodiment, Southernblot analysis of digested DNA from individual transposon mutants may beused to verify transposon insertion. In another embodiment, sequenceanalysis, PCR and/or hybridization may be utilized to determinetransposon insertion. Mutations may also be elicited usingethylmethanesulfonate (EMS) or radiation. In another embodiment,mutagenesis with chemical agents may be used. Such chemical mutagens maycomprise, in other embodiments, chemicals that affect nonreplicating DNAsuch as HNO2 and NH2OH, as well as agents that affect replicating DNAsuch as acridine dyes, which have been shown to cause frameshiftmutations. Methods for creating mutants using radiation or chemicalagents are well known in the art, and any method may be utilized for themethods of this invention (see, for example, Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol. 36, 227 (1992).

In one embodiment, DNA is synthesized chemically from the fournucleotides in whole or in part by methods known in the art. Suchmethods include those described in Caruthers (1985; Science230:281-285). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together (see, generally, Sambrook et al. (1989; MolecularCloning—A Laboratory Manual, 2nd Edition. Cold Spring Habour LaboratoryPress, New York)). In another embodiment, inactivating mutations areprepared from wild-type DNA by site-directed mutagenesis (see, forexample, Zoller et al. (1982; DNA. 1984 December; 3(6):479-88); Zoller(1983); and Zoller (1984; DNA. 1984 December; 3(6):479-88); McPherson(1991; Directed Mutagenesis: A Practical Approach. Oxford UniversityPress, NY)). The DNA obtained can be amplified by methods known in theart. One suitable method is the polymerase chain reaction (PCR) methoddescribed in Saiki et al. (1988; Science. 1988 Jan. 29;239(4839):487-491), Mullis et al., U.S. Pat. No. 4,683,195, and Sambrooket al. (1989).

In one embodiment, the present invention provides a method of impedingthe establishment of a latent HSV infection in a subject, comprising thestep of contacting the subject with a mutant HSV strain, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the latent HSV infection that isprevented follows primary HSV infection. In another embodiment, thesubject has been infected with HSV before vaccination. In anotherembodiment, the subject is at risk for HSV infection. In anotherembodiment, whether or not the subject has been infected with HSV at thetime of vaccination, vaccination by a method of the present invention isefficacious in protecting a subject against latent HSV infection,following primary HSV infection.

In one embodiment, the present invention provides a method of inhibitingan HSV flare in a subject, comprising the step of contacting the subjectwith a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein. In anotherembodiment, the flare that is prevented follows exposure of the subjectto HSV. In another embodiment, the subject has been infected with HSVbefore vaccination. In another embodiment, the subject is at risk forHSV infection. In another embodiment, whether or not the subject hasbeen infected with HSV at the time of vaccination, vaccination by amethod of the present invention is efficacious in protecting a subjectagainst a formation of a flare, following an exposure of the subject toHSV.

In one embodiment, the present invention provides a method of protectinga subject against an HSV flare, comprising the step of contacting thesubject with a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein. In anotherembodiment, the flare that is prevented follows exposure of the subjectto HSV. In another embodiment, the subject has been infected with HSVbefore vaccination. In another embodiment, the subject is at risk forHSV infection. In another embodiment, whether or not the subject hasbeen infected with HSV at the time of vaccination, vaccination by amethod of the present invention is efficacious in protecting a subjectagainst a formation of a flare, following exposure of the subject toHSV.

In one embodiment, the present invention provides a method of reducingthe incidence of an HSV flare, comprising the step of contacting thesubject with a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein. In anotherembodiment, the flare that is prevented follows exposure of the subjectto HSV. In another embodiment, the subject has been infected with HSVbefore vaccination. In another embodiment, the subject is at risk forHSV infection. In another embodiment, whether or not the subject hasbeen infected with HSV at the time of vaccination, vaccination by amethod of the present invention is efficacious in reducing the incidenceof a flare, following exposure of the subject to HSV.

In one embodiment, the present invention provides a method of inhibitingHSV recurrence in a subject, comprising the step of contacting thesubject with a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein. In anotherembodiment, the recurrence that is prevented follows exposure of thesubject to HSV. In another embodiment, the subject has been infectedwith HSV before vaccination. In another embodiment, the subject is atrisk for HSV infection. In another embodiment, whether or not thesubject has been infected with HSV at the time of vaccination,vaccination by a method of the present invention is efficacious inprotecting a subject against a recurrence, following an exposure of thesubject to an HSV.

In one embodiment, the present invention provides a method of reducingthe incidence of HSV recurrence, comprising the step of contacting thesubject with a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein. In anotherembodiment, the recurrence that is prevented follows exposure of thesubject to HSV. In another embodiment, the subject has been infectedwith HSV before vaccination. In another embodiment, the subject is atrisk for HSV infection. In another embodiment, whether or not thesubject has been infected with HSV at the time of vaccination,vaccination by a method of the present invention is efficacious inreducing the incidence of a recurrence, following exposure of thesubject to HSV.

“Flare” or “recurrence” refers, in one embodiment, to reinfection (inone embodiment, of skin tissue) following latent neuronal HSV infection.In another embodiment, the terms refer to reactivation of HSV after alatency period. In another embodiment, the terms refer to symptomaticHSV lesions following a non-symptomatic latency period.

In another embodiment, the present invention provides a method ofsuppressing HSV-1 infection in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutant HSVstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the mutant HSV strain is a mutant HSV-1strain. In another embodiment, the mutant HSV strain is a mutant HSV-2strain.

In another embodiment, the present invention provides a method ofsuppressing HSV-2 infection in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutant HSVstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the mutant HSV strain is a mutant HSV-1strain. In another embodiment, the mutant HSV strain is a mutant HSV-2strain.

According to any of the methods of the present invention and in oneembodiment, the subject is human. In another embodiment, the subject ismurine, which in one embodiment is a mouse, and, in another embodimentis a rat. In another embodiment, the subject is canine, feline, bovine,or porcine. In another embodiment, the subject is mammalian. In anotherembodiment, the subject is any organism susceptible to infection by HSV.

In one embodiment, the subject is infected by HSV, while in anotherembodiment, the subject is at risk for infection by HSV. In oneembodiment, a subject at risk for HSV infection is a neonate. In anotherembodiment, a subject at risk for HSV infection is immunocompromised. Inanother embodiment, a subject at risk for HSV infection is elderly. Inanother embodiment, a subject at risk for HSV infection is animmunocompromised neonate or an immunocompromised elderly subject.

In another embodiment, the present invention provides a method ofprotecting a subject against formation of a zosteriform lesion or ananalogous outbreak in a human subject, comprising the step of contactingthe subject with a mutant HSV strain, wherein the mutant strain containsan inactivating mutation in a Us8 gene encoding a gE protein.

In another embodiment, the present invention provides a method ofimpeding formation of an HSV zosteriform lesion or an analogous outbreakin a human subject, comprising the step of contacting the subject with amutant HSV strain, wherein the mutant strain contains an inactivatingmutation in a Us8 gene encoding a gE protein.

In another embodiment, the zosteriform lesion or analogous outbreak thatis impeded follows exposure of the subject to HSV. In anotherembodiment, the subject has been infected with HSV before vaccination.In another embodiment, the subject is at risk for HSV infection. Inanother embodiment, whether or not the subject has been infected withHSV at the time of vaccination, vaccination by a method of the presentinvention is efficacious in impeding formation of a zosteriform lesionor analogous outbreak, following an exposure of the subject to an HSV.

In another embodiment, the present invention provides a method ofimpeding HSV zosteriform spread or an analogous condition in a humansubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein. In another embodiment, the zosteriformspread or analogous condition that is impeded follows exposure of thesubject to HSV. In another embodiment, the subject has been infectedwith HSV before vaccination. In another embodiment, the subject is atrisk for HSV infection. In another embodiment, whether or not thesubject has been infected with HSV at the time of vaccination,vaccination by a method of the present invention is efficacious inimpeding formation of a zosteriform spread or analogous condition,following exposure of the subject to HSV.

“Zosteriform” refers, in one embodiment, to skin lesions characteristicof an HSV infection, particularly during reactivation infection, which,in one embodiment, begin as a rash and follow a distribution neardermatomes, commonly occurring in a strip or belt-like pattern. In oneembodiment, the rash evolves into vesicles or small blisters filled withserous fluid. In one embodiment, zosteriform lesions form in mice as aresult of contact with HSV. In another embodiment, zosteriform lesionsform in humans as a result of contact with HSV.

“Zosteriform spread” refers, in one embodiment, to an HSV infection thatspreads from the ganglia to secondary skin sites within the dermatome.In another embodiment, the term refers to spread within the samedermatome as the initial site of infection. In another embodiment, theterm refers to any other definition of “zosteriform spread” known in theart. “Outbreak”, in another embodiment, refers to a sudden increase insymptoms of a disease or in the spread or prevalence of a disease, andin one embodiment, refers to a sudden increase in zosteriform lesions,while in another embodiment, “outbreak” refers to a sudden eruption ofzosteriform lesions.

In one embodiment, the present invention provides a method of impedingthe formation of a dermatome lesion or an analogous condition in a humansubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein. In another embodiment, the dermatomelesion or analogous condition that is impeded follows exposure of thesubject to HSV. In one embodiment, dermatome lesions form in humans as aresult of contact with HSV. In another embodiment, dermatome lesions inhumans most often develop when the virus reactivates from latency in theganglia and in one embodiment, spreads down nerves, in one embodiment,causing a recurrent infection. In another embodiment, dermatome lesionsform in mice as a result of contact with HSV. In another embodiment, thesubject has been infected with HSV before vaccination. In anotherembodiment, the subject is at risk for HSV infection. In anotherembodiment, whether or not the subject has been infected with HSV at thetime of vaccination, vaccination by a method of the present invention isefficacious in impeding the formation of a dermatome lesion or analogouscondition, following exposure of the subject to HSV.

In one embodiment, vaccination with gE-null HSV strains of the presentinvention protects against latent HSV infection (Example 5) andformation of zosteriform and dermatome lesions (Examples 4 and 20) aftersubsequent infection with virulent HSV. In another embodiment, thevaccination protects against disease caused by or associated with latentHSV infection. In another embodiment, the vaccination does not itselfcause significant disease (Examples 2 and 19). In another embodiment,the vaccination protects against death, vaginal disease and recurrentinfection (Example 30 and 31). In another

“Virulent HSV” refers, in one embodiment, to a naturally occurring HSVstrain. In another embodiment, the term refers to an HSV strain capableof causing infection. In another embodiment, the term refers to an HSVstrain capable of establishing latent infection.

In another embodiment, the present invention provides a method ofimpeding neuronal spread of an HSV in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the neuronal spread that is impededfollows exposure of the subject to HSV. In another embodiment, thesubject has been infected with HSV before vaccination. In anotherembodiment, the subject is at risk for HSV infection. In anotherembodiment, whether or not the subject has been infected with HSV at thetime of vaccination, vaccination by a method of the present invention isefficacious in impeding neuronal viral spread, following an exposure ofthe subject to HSV.

Methods of measuring neuronal HSV spread are well known in the art, andinclude, in one embodiment, determination of the presence and extent ofsecondary dermatome lesion (Example 2). Other embodiments of methods formeasuring viral spread are described, for example, in Labetoulle M etal. (Neuronal propagation of HSV1 from the oral mucosa to the eye.Invest Ophthalmol Vis Sci. 2000 August; 41(9):2600-6) and Thompson K Aet al. (Herpes simplex replication and dissemination is not increased bycorticosteroid treatment in a rat model of focal Herpes encephalitis. JNeurovirol. 2000 February; 6(1):25-32).

In one embodiment, the present invention provides a method of reducingthe incidence of herpetic ocular disease in a subject, comprising thestep of contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by the HSV.

In one embodiment, the present invention provides a method of reducingthe severity of herpetic ocular disease in a subject, comprising thestep of contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by HSV. Inanother embodiment, the vaccine strain is from a different species fromthe challenge strain. In another embodiment, the vaccine strain is ofthe same species as the challenge strain.

In another embodiment, the present invention provides a method ofreducing the incidence of an HSV-1 corneal infection, herpes keratitisor any other herpetic ocular disease in a subject, the method comprisingthe step of administering to said subject a mutant strain of HSV of thepresent invention, thereby reducing an incidence of an HSV-1 cornealinfection or herpes keratitis in a subject. In another embodiment,administering to said subject a mutant strain of HSV of the presentinvention elicits an immune response against the HSV-1.

Methods for determining the presence and extent of herpetic oculardisease, corneal infection, and herpes keratitis are well known in theart, and are described, for example, in Labetoulle M et al. (Neuronalpropagation of HSV1 from the oral mucosa to the eye. Invest OphthalmolVis Sci. 2000 August; 41(9):2600-6) and Majumdar S i (Dipeptidemonoester ganciclovir prodrugs for treating HSV-1-induced cornealepithelial and stromal keratitis: in vitro and in vivo evaluations. JOcul Pharmacol Ther. 2005 December; 21(6):463-74).

In one embodiment, the present invention provides a method of reducingthe incidence of a genital ulcer disease in a subject, comprising thestep of contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by HSV

In one embodiment, the present invention provides a method of reducingthe severity of genital ulcer disease in a subject, comprising the stepof contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by HSV.

In one embodiment, genital ulcer disease is characterized by ulcerativelesions on the genitals. Methods for determining the presence and extentof genital ulcer disease are well known in the art.

In one embodiment, the present invention provides a method of reducingthe incidence of HSV-1-mediated encephalitis in a subject, comprisingthe step of contacting the subject with a mutant strain of HSV, whereinthe mutant strain contains an inactivating mutation in a Us8 geneencoding a gE protein. “HSV-1 encephalitis” refers, in one embodiment,to encephalitis caused by HSV-1. In another embodiment, the term refersto encephalitis associated with HSV-1. In another embodiment, the termrefers to any other type of HSV-1-mediated encephalitis known in theart. In another embodiment, the subject is infected by HSV. In anotherembodiment, the subject is at risk of infection by HSV. In anotherembodiment, the vaccine strain is from a different species from thechallenge strain. In another embodiment, the vaccine strain is of thesame species as the challenge strain.

In another embodiment, the present invention provides a method ofreducing the incidence of HSV-2-mediated encephalitis in a subject,comprising the step of contacting the subject with a mutant strain ofHSV, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein. “HSV-2 encephalitis” refers, in oneembodiment, to encephalitis caused by HSV-2. In another embodiment, theterm refers to encephalitis associated with HSV-2. In anotherembodiment, the term refers to any other type of HSV-2-mediatdencephalitis known in the art. In another embodiment, the subject isinfected by HSV. In another embodiment, the subject is at risk ofinfection by HSV

In one embodiment, the present invention provides a method of reducingthe severity of herpes-mediated encephalitis in a subject, comprisingthe step of contacting the subject with a mutant strain of HSV, whereinthe mutant strain contains an inactivating mutation in a Us8 geneencoding a gE protein. In one embodiment, the subject is infected byHSV. In another embodiment, the subject is at risk of infection by HSV.

In one embodiment, the herpes-mediated encephalitis treated or preventedby a method of the present invention is a focal herpes encephalitis. Inanother embodiment, the herpes-mediated encephalitis is a neonatalherpes encephalitis. In another embodiment, the herpes-mediatedencephalitis is any other type of herpes-mediated encephalitis known inthe art.

In one embodiment, the present invention provides a method of reducingthe incidence of disseminated HSV infection in a subject, comprising thestep of contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In one embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by the HSV.

In one embodiment, the present invention provides a method of reducingthe severity of disseminated HSV infection in a subject, comprising thestep of contacting the subject with a mutant strain of HSV, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the subject is infected by HSV. Inanother embodiment, the subject is at risk of infection by HSV.

In one embodiment, the present invention provides a method of reducingthe incidence of a neonatal HSV-1 infection in an offspring of asubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant HSV strain contains an inactivating mutationin a Us8 gene encoding a gE protein. In one embodiment, the offspring iscontacted the subject with the mutant HSV strain. In another embodiment,the subject is infected by HSV. In another embodiment, the subject is atrisk of infection by HSV.

In one embodiment, the present invention provides a method of reducingthe incidence of a neonatal HSV-2 infection in an offspring of asubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant HSV strain contains an inactivating mutationin a Us8 gene encoding a gE protein. In one embodiment, the subject isinfected by HSV. In another embodiment, the subject is at risk ofinfection by HSV.

In one embodiment, the present invention provides a method of reducingthe transmission of an HSV-1 infection from a subject to an offspringthereof, the method comprising the step of contacting the subject with amutant HSV strain, wherein the mutant HSV strain contains aninactivating mutation in a Us8 gene encoding a gE protein.

In one embodiment, the present invention provides a method of reducingthe transmission of an HSV-2 infection from a subject to an offspringthereof, the method comprising the step of contacting the subject with amutant HSV strain, wherein the mutant HSV strain contains aninactivating mutation in a Us8 gene encoding a gE protein.

In one embodiment, the present invention provides a method of reducingHIV-1 transmission to an offspring, the method comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutant HSVstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. As is known in the art, HSV-2 infection increases HIV-1 viralshedding in genital secretions (Nagot N et al. Reduction of HIV-1 RNAlevels with therapy to suppress herpes simplex virus. N Engl J Med. 2007Feb. 22; 356(8):790-9). Thus, methods of the present invention ofinhibiting HSV-2 infection are also believed to be efficacious forreducing HIV-1 transmission to an offspring. In another embodiment, themutant HSV strain is an HSV-1 strain. In another embodiment, the mutantHSV strain is an HSV-2 strain.

In one embodiment, the present invention provides a method of reducingHIV-1 transmission to a sexual partner, the method comprising the stepof contacting the subject with a mutant HSV strain, wherein the mutantHSV strain contains an inactivating mutation in a Us8 gene encoding a gEprotein. As is known in the art, HSV-2 infection increases HIV-1 viralshedding in genital secretions. Thus, methods of the present inventionof inhibiting HSV-2 infection are also believed to be efficacious forreducing HIV-1 transmission to a sexual partner. In another embodiment,the mutant HSV strain is an HSV-1 strain. In another embodiment, themutant HSV strain is an HSV-2 strain.

In one embodiment, the present invention provides a method of reducingsusceptibility to HIV-1, the method comprising the step of contactingthe subject with a mutant HSV strain, wherein the mutant HSV straincontains an inactivating mutation in a Us8 gene encoding a gE protein.As is known in the art, HSV-2 infection increases HIV-1 replication(Ouedraogo A et al Impact of suppressive herpes therapy on genital HIV-1RNA among women taking antiretroviral therapy: a randomized controlledtrial. AIDS. 2006 Nov. 28; 20(18):2305-13). Thus, methods of the presentinvention of inhibiting HSV-2 infection are also believed to beefficacious for reducing susceptibility to HIV-1. In another embodiment,the mutant HSV strain is an HSV-1 strain. In another embodiment, themutant HSV strain is an HSV-2 strain.

In one embodiment, the present invention provides a method of reducingthe severity of a neonatal HSV infection in an offspring of a subject,comprising the step of contacting the subject with a mutant strain ofHSV, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein. In another embodiment, the subject isinfected by HSV. In another embodiment, the subject is at risk ofinfection by HSV.

In one embodiment, the present invention provides a method of reducingthe incidence of a disease, disorder, or symptom associated with orsecondary to a herpes-mediated encephalitis in a subject, comprising thestep of contacting the subject with a mutant HSV strain, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein.

In one embodiment, the present invention provides a method of treating adisease, disorder, or symptom associated with or secondary to aherpes-mediated encephalitis in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein, thereby treating a disease, disorder, or symptom associatedwith or secondary to a herpes-mediated encephalitis in a subject.

In one embodiment, the disease, disorder, or symptom is fever. Inanother embodiment, the disease, disorder, or symptom is headache. Inanother embodiment, the disease, disorder, or symptom is stiff neck. Inanother embodiment, the disease, disorder, or symptom is seizures. Inanother embodiment, the disease, disorder, or symptom is partialparalysis. In another embodiment, the disease, disorder, or symptom isstupor. In another embodiment, the disease, disorder, or symptom iscoma. In another embodiment, the disease, disorder, or symptom is anyother disease, disorder, or symptom known in the art that is associatedwith or secondary to herpes-mediated encephalitis.

In another embodiment, “symptoms” may be any manifestation of a HSVinfection, including, but not limited to blisters, ulcerations, orlesions on the urethra, cervix, upper thigh, and/or anus in women and onthe penis, urethra, scrotum, upper thigh, and anus in men, inflammation,swelling, fever, flu-like symptoms, sore mouth, sore throat,pharyngitis, pain, blisters on tongue, mouth or lips, ulcers, coldsores, neck pain, enlarged lymph nodes, reddening, bleeding, itching,dysuria, headache, muscle pain, etc., or a combination thereof.

In another embodiment, the disease, disorder, or symptom is fever. Inanother embodiment, the disease, disorder, or symptom is headache. Inanother embodiment, the disease, disorder, or symptom is stiff neck. Inanother embodiment, the disease, disorder, or symptom is seizures. Inanother embodiment, the disease, disorder, or symptom is partialparalysis. In another embodiment, the disease, disorder, or symptom isstupor. In another embodiment, the disease, disorder, or symptom iscoma. In another embodiment, the disease, disorder, or symptom is anyother disease, disorder, or symptom known in the art that is associatedwith or secondary to a herpes-mediated encephalitis.

In one embodiment, a mutant HSV-1 strain of the present inventionprotects a subject against infection and disorders and symptomsassociated with infection with wild-type HSV-1. In another embodiment,the disorders and symptoms include herpes labialis (cold sores or feverblisters). In another embodiment, the disorders and symptoms includeHSV-mediated cornea disease. In another embodiment, the disorders andsymptoms include herpes-mediated retinitis. In another embodiment, thedisorders and symptoms include herpes-mediated encephalitis. In anotherembodiment, the disorders and symptoms include HSV-1-mediated genitalulcer disease. In another embodiment, a mutant HSV-1 strain of thepresent invention provides substantial protection against HSV-1infection and partial protection against one or more symptoms associatedwith HSV-2 infection. In another embodiment, these HSV-2 symptomsinclude the symptoms described hereinabove.

Methods of determining the presence and severity of herpes-mediatedencephalitis are well known in the art, and are described, for example,in Bonkowsky J L et al. (Herpes simplex virus central nervous systemrelapse during treatment of infantile spasms with corticotropin.Pediatrics. 2006 May; 117(5):e1045-8) and Khan O A et al. (Herpesencephalitis presenting as mild aphasia: case report. BMC Fam Pract.2006 Mar. 24; 7:22).

In one embodiment, the present invention provides a method of treating adisease, disorder, or symptom associated with an HSV infection in asubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein.

In one embodiment, the present invention provides a method of reducingthe incidence of a disease, disorder, or symptom associated with an HSVinfection in a subject, comprising the step of contacting the subjectwith a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein.

In one embodiment, the present invention provides a method of treating adisease, disorder, or symptom secondary to an HSV infection in asubject, comprising the step of contacting the subject with a mutant HSVstrain, wherein the mutant strain contains an inactivating mutation in aUs8 gene encoding a gE protein.

In one embodiment, the present invention provides a method of reducingthe incidence of a disease, disorder, or symptom secondary to an HSVinfection in a subject, comprising the step of contacting the subjectwith a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein.

In one embodiment, the disease, disorder, or symptom secondary to an HSVinfection is oral lesions. In another embodiment, the disease, disorder,or symptom is genital lesions. In another embodiment, the disease,disorder, or symptom is oral ulcers. In another embodiment, the disease,disorder, or symptom is genital ulcers. In another embodiment, thedisease, disorder, or symptom is fever. In another embodiment, thedisease, disorder, or symptom is headache. In another embodiment, thedisease, disorder, or symptom is muscle ache. In another embodiment, thedisease, disorder, or symptom is swollen glands in the groin area. Inanother embodiment, the disease, disorder, or symptom is painfulurination. In another embodiment, the disease, disorder, or symptom isvaginal discharge. In another embodiment, the disease, disorder, orsymptom is blistering. In another embodiment, the disease, disorder, orsymptom is flu-like malaise. In another embodiment, the disease,disorder, or symptom is keratitis. In another embodiment, the disease,disorder, or symptom is herpetic whitlow. In another embodiment, thedisease, disorder, or symptom is Bell's palsy. In another embodiment,the disease, disorder, or symptom is herpetic erythema multiforme. Inanother embodiment, the disease, disorder, or symptom is a lower backsymptom (e.g. numbness, tingling of the buttocks or the area around theanus, urinary retention, constipation, and impotence). In anotherembodiment, the disease, disorder, or symptom is a localized eczemaherpeticum. In another embodiment, the disease, disorder, or symptom isa disseminated eczema herpeticum. In another embodiment, the disease,disorder, or symptom is a herpes gladiatorum. In another embodiment, thedisease, disorder, or symptom is a herpetic sycosis. In anotherembodiment, the disease, disorder, or symptom is an esophageal symptom(e.g. difficulty swallowing or burning, squeezing throat pain whileswallowing, weight loss, pain in or behind the upper chest whileswallowing). In another embodiment, the disease, disorder, or symptom isany other disease, disorder, or symptom is known in the art.

The HSV infection treated or ameliorated by methods and compositions ofthe present invention is, in one embodiment, a genital HSV infection. Inanother embodiment, the HSV infection is an oral HSV infection. Inanother embodiment, the HSV infection is an ocular HSV infection. Inanother embodiment, the HSV infection is a dermatologic HSV infection.

In one embodiment, the HSV infection is an HSV-2 infection. In anotherembodiment, the HSV is an HSV-1 infection. In another embodiment, theHSV infection is any other type of HSV infection known in the art.

In one embodiment, the present invention provides a method of inducingrapid clearance of an HSV-1 infection in a subject, comprising the stepof contacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the mutant HSV strain is a mutant HSV-1strain. In another embodiment, the mutant HSV strain is a mutant HSV-2strain.

In one embodiment, the present invention provides a method of inducingrapid clearance of an HSV-2 infection in a subject, comprising the stepof contacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein. In another embodiment, the mutant HSV strain is a mutant HSV-1strain. In another embodiment, the mutant HSV strain is a mutant HSV-2strain.

In one embodiment, the present invention provides a method of inducingan anti-HSV immune response in a subject, comprising the step ofcontacting the subject with a mutant HSV strain, wherein the mutantstrain contains an inactivating mutation in a Us8 gene encoding a gEprotein, thereby inducing an anti-HSV immune response in a subject. Inanother embodiment, the mutant HSV strain is a mutant HSV-1 strain. Inanother embodiment, the mutant HSV strain is a mutant HSV-2 strain.

In one embodiment, the present invention provides a method of inducingan anti-HSV neutralizing antibody response in a subject, comprising thestep of contacting the subject with a mutant HSV strain, wherein themutant strain contains an inactivating mutation in a Us8 gene encoding agE protein. In another embodiment, the mutant HSV strain is a mutantHSV-1 strain. In another embodiment, the mutant HSV strain is a mutantHSV-2 strain. In another embodiment, no antibody response to gD-2 isdetected after the first immunization with HSV-2ΔgE(gfp) as shown inExample 25 herein.

In one embodiment, a first immunization with the live virus vaccineHSV-2ΔgE(gfp) results in an antibody response. In another embodiment, asecond administration or immunization with the live virus vaccineprovided herein results in a high titer and more pronounced antibodyresponse as compared to the first immunization (see Example 26, herein).

In another embodiment, a second administration or immunization with thelive virus vaccine provided herein produces high titers of anti-gC-2 andanti-gD-2 antibodies than one immunization alone (see Example 31,herein).

In one embodiment, the present invention provides a method of inhibitingHSV labialis in a subject, comprising the step of vaccinating thesubject against an HSV by a method of the present invention.

In one embodiment, the examples of the present invention provideexperimental support for a method of vaccinating against HSV infectionby contacting the subject with a mutant strain of HSV, containing one ormore inactivating mutations.

In another embodiment, the present invention provides a method ofinhibiting HSV labialis in a subject, comprising the step of impeding anHSV infection in the subject by a method of the present invention.

In one embodiment, the immune response induced by methods andcompositions of the present invention is a cellular immune response. Inanother embodiment, the immune response comprises a CD8+ cytotoxic Tlymphocyte (CTL) response. In another embodiment, the immune responsecomprises a CD4+ helper T cell response. In another embodiment, theimmune response comprises a humoral immune response. In one embodiment,an immune response refers to an in vivo or in vitro reaction in responseto a challenge by an immunogen. In one embodiment, an immune response isexpressed by antibody production, cell-mediated immunity, immunologictolerance, or a combination thereof.

The route of administration of the mutant strains in the methods of thepresent invention is, in one embodiment, epidermal. In anotherembodiment, the mutant strain is administered by epidermal scarificationor scratching. In another embodiment, the mutant strain is administeredintramuscularly. In another embodiment, the mutant strain isadministered subcutaneously. In another embodiment, the mutant strain isadministered intranasally. In another embodiment, the mutant strain isadministered transdermally. In another embodiment, the mutant strain isadministered intravaginally. In another embodiment, the mutant strain isadministered transmucosally, which in one embodiment, isintra-respiratory mucosally. In another embodiment, the mutant strain isadministered intranasally. In another embodiment, the mutant strain isadministered in an aerosol. In another embodiment, the mutant strain isadministered via any other route known in the art.

In one embodiment, the inactivating mutation in the gE-encoding gene ofHSV strains as described in the methods and compositions of the presentinvention is a deletion mutation. In another embodiment, theinactivating mutation is an insertion mutation. In another embodiment,the inactivating mutation is a substitution mutation. In anotherembodiment, the inactivating mutation is a gE-null mutation. In anotherembodiment, the inactivating mutation is any other type of mutationknown in the art.

In one embodiment, the inactivating mutation in theglycoprotein-encoding gene of HSV strains as described in the methodsand compositions of the present invention is a deletion mutation. Inanother embodiment, the inactivating mutation is an insertion mutation.In another embodiment, the inactivating mutation is a substitutionmutation. In another embodiment, the inactivating mutation is a nullmutation. In another embodiment, the inactivating mutation is any othertype of mutation known in the art. In one embodiment, the insertion,deletion or substitution mutation comprises an insertion, deletion orsubstitution of a single amino acid, while in another embodiment, itcomprises an insertion, deletion or substitution of 1-5 amino acids,1-10 amino acids, 5-20 amino acids, 10-50 amino acids, 25-100 aminoacids, 100-500 amino acids, 300-400 amino acids, 200-1000 amino acids,or 1000 or more amino acids.

In one embodiment, the present invention provides an isolated mutantHSV-1 strain comprising a first inactivating mutation in a gene encodinga gE protein and a second inactivating mutation. In another embodiment,the gene encoding a gE protein is a Us8 gene. In another embodiment, themutation is a gE-null mutation. In one embodiment, an isolated mutantHSV-1 strain as described in the methods and compositions of the presentinvention further comprises one or more additional mutations, which inone embodiment are inactivating mutations. In another embodiment, thesecond or additional inactivating mutation is in a Us7 gene. In anotherembodiment, the second or additional inactivating mutation is in a Us9gene. In another embodiment, the second inactivating mutation is in anygene which confers neurovirulence. In another embodiment, the secondinactivating mutation is in any gene required for virus entry into ahost cell. In another embodiment, the second inactivating mutation is ina host shut-off gene. In another embodiment, the second inactivatingmutation is in the thymidine kinase gene. In another embodiment, thesecond inactivating mutation is in any other HSV-1 gene known in theart. In another embodiment, the isolated mutant HSV-1 strain containsinactivating mutations in a gene encoding a gE protein, a Us7 gene, anda Us9 gene. In another embodiment, an isolated mutant HSV-1 strain asdescribed in the methods and compositions of the present inventionfurther comprises an additional mutation in a gene encoding a gDprotein.

In one embodiment, the present invention provides an isolated mutantHSV-2 strain comprising a first inactivating mutation in a gene encodinga gE protein and a second inactivating mutation. In another embodiment,the gene encoding a gE protein is a Us8 gene. In another embodiment, themutation is a gE-null mutation. In one embodiment, an isolated mutantHSV-2 strain as described in the methods and compositions of the presentinvention further comprises one or more additional mutations, which inone embodiment are inactivating mutations. In another embodiment, thesecond or additional inactivating mutation is in a Us7 gene. In anotherembodiment, the second or additional inactivating mutation is in a Us9gene. In another embodiment, the second inactivating mutation is in anygene which confers neurovirulence. In another embodiment, the secondinactivating mutation is in any gene required for virus entry into ahost cell. In another embodiment, the second inactivating mutation is ina host shut-off gene. In another embodiment, the second inactivatingmutation is in the thymidine kinase gene. In another embodiment, thesecond inactivating mutation is in any other HSV-2 gene known in theart. In another embodiment, the isolated mutant HSV-2 strain containsinactivating mutations in a gene encoding a gE protein, a Us7 gene, anda Us9 gene. In another embodiment, an isolated mutant HSV-2 strain asdescribed in the methods and compositions of the present inventionfurther comprises an additional mutation in a gene encoding a gDprotein.

In one embodiment, the Us7 gene that is mutated is highly conservedamongst alpha-herpesviruses. In another embodiment, the Us7 gene that ismutated is required for anterograde spread of the virus. In anotherembodiment, the Us7 gene that is mutated is required for retrogradespread of the virus.

In one embodiment, the Us9 gene that is mutated is highly conservedamongst alpha-herpesviruses. In another embodiment, the Us9 gene that ismutated is required for anterograde spread of the virus. In anotherembodiment, the Us9 gene that is mutated is required for retrogradespread of the virus.

In one embodiment, the mutation in Us7 and/or Us9 is an inactivatingmutation. In another embodiment, the mutation is a deletion mutation. Inanother embodiment, the mutation is an insertion mutation. In anotherembodiment, the mutation is a substitution mutation. In anotherembodiment, the mutation is any other type of mutation known in the art.

In one embodiment, a mutant strain of the present invention furthercomprises an additional inactivating mutation in a gene encoding amembrane protein not required for virus entry. In one embodiment, a geneencoding a membrane protein not required for virus entry is Us7 gene orUs9 gene. In another embodiment, a gene encoding a membrane protein notrequired for virus entry is Us5, Us4, UL53, or UL10. In anotherembodiment, a mutant strain of the present invention further comprisesan additional inactivating mutation in a gene encoding a membraneprotein required for virus entry, which in one embodiment, is Us6.

In one embodiment, the additional gene that is mutated is highlyconserved amongst alpha-herpesviruses. In another embodiment, theadditional gene that is mutated is required for anterograde spread ofthe virus. In another embodiment, the additional gene that is mutated isrequired for retrograde spread of the virus.

In one embodiment, the additional gene that is mutated is a virionmembrane protein. In one embodiment, the additional gene is a virionmembrane protein not required, or non-essential, for virus entry. Inanother embodiment, the membrane protein is a glycoprotein. In anotherembodiment, the additional gene is glycoprotein J. In anotherembodiment, the additional gene is glycoprotein G. In anotherembodiment, the additional gene is glycoprotein K. In anotherembodiment, the additional gene is glycoprotein M. In anotherembodiment, the additional gene is selected from glycoproteins J, G, K,and M.

In one embodiment, the additional mutation is introduced to enhanceinhibition of anterograde spread of the mutant HSV-1 strain. In anotherembodiment, the additional mutation is required, in combination with agE mutation, to confer inhibition of anterograde spread of the mutantHSV-1 strain. In another embodiment, the gE mutation is insufficient toconfer inhibition of anterograde spread of the mutant HSV-1 strain. Inanother embodiment, the additional mutation is sufficient, in theabsence of a gE mutation, to confer inhibition of anterograde spread ofthe mutant HSV-1 strain.

In one embodiment, the additional mutation is introduced to enhanceinhibition of anterograde spread of the mutant HSV-2 strain. In anotherembodiment, the additional mutation is required, in combination with agE mutation, to confer inhibition of anterograde spread of the mutantHSV-2 strain. In another embodiment, the gE mutation is insufficient toconfer inhibition of anterograde spread of the mutant HSV-2 strain. Inanother embodiment, the additional mutation is sufficient, in theabsence of a gE mutation, to confer inhibition of anterograde spread ofthe mutant HSV-2 strain.

In one embodiment, the additional mutation is introduced to enhanceattenuation of virulence in the HSV-1 or HSV-2 strain or both. Inanother embodiment, the additional mutation is required, in combinationwith a gE mutation, to attenuate virulence.

In one embodiment, the additional gene that is mutated is a virionmembrane protein. In another embodiment, the additional gene is a virionmembrane protein required for virus entry. In another embodiment, theadditional gene is glycoprotein B. In another embodiment, the additionalgene is glycoprotein D. In another embodiment, the additional gene isglycoprotein H. In another embodiment, the additional gene isglycoprotein L.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention is replication-competent. Inanother embodiment, the mutant HSV strain as described in the methodsand compositions of the present invention is replication-competent inskin tissue of the subject. In another embodiment, the mutant strain isreplication-competent in skin cell of the subject. In anotherembodiment, the mutant strain is replication-competent in skin tissue ofthe species to which the subject belongs. In another embodiment, themutant strain is replication-competent in a cell line derived from skintissue of the subject's species. In another embodiment, the mutantstrain is replication-competent in a culture of skin cells of thesubject's species. In another embodiment, the mutant strain isreplication-competent in a cell line derived from a skin cell of thesubject's species.

“Replication competent” refers, in one embodiment, to an ability toreplicate. In another embodiment, the term includes strains that exhibitimpaired but still detectable levels of replication. In anotherembodiment, the term refers to a strain that exhibits measurablereplication.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention is defective in its ability tospread from the site of inoculation to the dorsal root ganglia (DRG). Inone embodiment, the dorsal root ganglia contain the neuron cell bodiesof nerve fibres. In another embodiment, the mutant HSV strain isdefective in retrograde spread. In another embodiment, the mutant HSVstrain is impaired in retrograde spread. In another embodiment, themutant HSV strain is significantly impaired in retrograde spread. Inanother embodiment, the mutant HSV strain is impaired in retrogradespread but is replication-competent in skin.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention is defective in spread from DRG tothe skin. In another embodiment, the mutant HSV strain is defective inanterograde spread. In another embodiment, the mutant HSV strain isimpaired in anterograde spread. In another embodiment, the mutant HSVstrain is significantly impaired in anterograde spread. In anotherembodiment, the mutant HSV strain is impaired in anterograde spread butis replication-competent in skin. In another embodiment, the mutant HSVstrain is impaired in anterograde spread but is replication-competent atthe site of innoculation.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention is defective in spread from DRG tothe skin. In another embodiment, the mutant HSV strain is defective inretrograde spread. In another embodiment, the mutant HSV strain isimpaired in retrograde spread. In another embodiment, the mutant HSVstrain is significantly impaired in retrograde spread. In anotherembodiment, the mutant HSV strain is impaired in retrograde spread butis replication-competent in skin. In another embodiment, the mutant HSVstrain is impaired in anterograde spread but is replication-competent atthe site of innoculation.

“DRG” refers, in one embodiment, to a neuronal cell body. In anotherembodiment, the term refers to any other definition of “DRG” used in theart.

In one embodiment, a mutant HSV strain of the present invention isreplication-defective, either in a particular tissue (e.g. in neuraltissue) or in general. Methods for measuring viral replication are wellknown in the art and include, in one embodiment, titering assays oftissue samples near a site of inoculation (Examples herein). In anotherembodiment, recovery of infectious virus from tissues near a site ofinoculation is utilized (Examples herein). Other embodiments asdescribed in the methods for measuring viral replication are described,for example, in Thi T N et al. (Rapid determination of antiviral drugsusceptibility of herpes simplex virus types 1 and 2 by real-time PCR.Antiviral Res. 2006 March; 69(3):152-7); Schang L M et al. (Roscovitine,a specific inhibitor of cellular cyclin-dependent kinases, inhibitsherpes simplex virus DNA synthesis in the presence of viral earlyproteins. J Virol. 2000 March; 74(5):2107-20); and Kennedy P G et al.(Replication of the herpes simplex virus type 1 RL1 mutant 1716 inprimary neuronal cell cultures—possible relevance to use as a viralvector. J Neurol Sci. 2000 Oct. 1; 179(S 1-2):108-14).

In one embodiment, a mutant strain as described in the methods andcompositions of the present invention is impaired in its spread inneural tissue of the subject. In another embodiment, the mutant strainis impaired in its spread in a culture of neural cells of the subject.In another embodiment, the mutant strain is impaired in its spread inneural tissue of the species to which the subject belongs. In anotherembodiment, the mutant strain is impaired in its spread in a cell linederived from neural tissue of the subject's species. In anotherembodiment, the mutant strain is impaired in its spread in a culture ofneural cells of the subject's species. In another embodiment, the mutantstrain is impaired in its spread in a cell line derived from a neuralcell of the subject's species.

In one embodiment, a mutant strain as described in the methods andcompositions of the present invention is impaired in its ability toenter neural tissue of the subject. In another embodiment, the mutantstrain is impaired in its ability to enter a culture of neural cells ofthe subject. In another embodiment, the mutant strain is impaired in itsability to enter neural tissue of the species to which the subjectbelongs. In another embodiment, the mutant strain is impaired in itsability to enter a cell line derived from neural tissue of the subject'sspecies. In another embodiment, the mutant strain is impaired in itsability to enter a culture of neural cells of the subject's species. Inanother embodiment, the mutant strain is impaired in its ability toenter a cell line derived from a neural cell of the subject's species.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention induces an anti-HSV immuneresponse. In another embodiment, the immune response impedes replicationof the HSV in the subject. In another embodiment, the immune responseimpedes neuronal spread of the HSV in the subject. In anotherembodiment, the immune response results in killing of HSV-infected cellsin the subject.

In one embodiment, the mutant HSV strain as described in the methods andcompositions of the present invention is a mutant HSV-1 strain. Inanother embodiment, the mutant HSV-1 strain confers protection againstan HSV-1 infection, spread, or a consequence thereof (e.g. zosteriformlesions or herpetic ocular disease). In another embodiment, the mutantHSV strain is a mutant HSV-2 strain. In another embodiment, the mutantHSV strain is HSV-2ΔgE or HSV-2ΔgE(gfp). In another embodiment, themutant HSV-2 strain confers protection against an HSV-2 infection,spread, or a consequence thereof (e.g. zosteriform lesions or herpeticocular disease). In another embodiment, the mutant HSV strain is anyother mutant HSV strain known in the art. In another embodiment, themutant HSV strain provided herein confers protection in a dose dependentmanner. In another embodiment, the mutant HSV strain provided hereinconfers protection to the DRG against high dose challenge with an HSV-2strain (see Example 28, herein).

In another embodiment, the disorders and symptoms include HSV infectionsin an immunocompromised subject, including subjects with HIV. In anotherembodiment, a mutant HSV-1 strain of the present invention prevents orinhibits transmission of genital HSV-1 from a vaccinated mother to hernewborn infant. In another embodiment, a mutant HSV strain of thepresent invention treats, suppresses, prevents or inhibits HSV inelderly subjects.

In one embodiment, a mutant HSV-2 strain of the present inventionprotects a subject against infection and disorders and symptomsassociated with infection with wild-type HSV-2. In another embodiment,the mutant HSV-2 strain prevents or inhibits transmission of genitalHSV-2 from the vaccinated mother to her newborn infant. In anotherembodiment, the mutant HSV-2 strain prevents or inhibits genital ulcerdisease. In another embodiment, the mutant HSV-2 strain providessubstantial protection against both HSV-2 and HSV-1 infection.

According to any of the methods of the invention, and in one embodiment,the infection is an HSV-1 infection. In another embodiment, theinfection is an HSV-2 infection.

According to any of the methods of the invention, and in one embodiment,the vaccine strain is from a different species from the strain againstwhich protection is conferred (“challenge strain”). In anotherembodiment, the vaccine strain is of the same species as the challengestrain.

In one embodiment, a vaccine as described in the methods andcompositions of the present invention protects a subject against achallenge with heterologous HSV. In another embodiment, the heterologouschallenge is a different strain of the same species. In anotherembodiment, in the case of a mutant HSV-1 vaccine strain, the vaccineconfers protection against a heterologous HSV-1 strain. In anotherembodiment, in the case of a mutant HSV-2 vaccine strain, the vaccineconfers protection against a heterologous HSV-2 strain. In anotherembodiment, the heterologous strain has an antigenic protein that issignificantly heterologous relative to the vaccine strain. In anotherembodiment, the antigenic protein is gD. In another embodiment, theantigenic protein is gB. In another embodiment, the antigenic protein isany other antigenic protein known in the art.

In one embodiment, the heterologous strain has a gD protein that issignificantly heterologous relative to the vaccine strain. In anotherembodiment, the gD protein of the heterologous strain shares 50%homology with the vaccine strain. In another embodiment, the homologyshared between the gD protein of the heterologous strain and the vaccinestrain is 55%. In another embodiment, the homology shared is 60%. Inanother embodiment, the homology shared is 65%. In another embodiment,the homology shared is 70%. In another embodiment, the homology sharedis 75%. In another embodiment, the homology shared is 80%. In anotherembodiment, the homology shared is 85%. In another embodiment, thehomology shared is 90%. In another embodiment, the homology shared is95%. In another embodiment, the homology shared is 98%. In anotherembodiment, the homology shared is greater than 98%.

In one embodiment, the heterologous strain has a gB protein that issignificantly heterologous relative to the vaccine strain. In anotherembodiment, the gB protein of the heterologous strain shares 50%homology with the vaccine strain. In another embodiment, the homologyshared between the gB protein of the heterologous strain and the vaccinestrain is 55%. In another embodiment, the homology shared is 60%. Inanother embodiment, the homology shared is 65%. In another embodiment,the homology shared is 70%. In another embodiment, the homology sharedis 75%. In another embodiment, the homology shared is 80%. In anotherembodiment, the homology shared is 85%. In another embodiment, thehomology shared is 90%. In another embodiment, the homology shared is95%. In another embodiment, the homology shared is 98%. In anotherembodiment, the homology shared is greater than 98%.

In one embodiment, the heterologous challenge strain is HSV-1 NS. Inanother embodiment, the heterologous challenge strain is HSV-1(F). Inanother embodiment, the heterologous challenge strain is HSV-1(17). Inanother embodiment, the heterologous challenge strain is any other HSV-1strain known in the art.

In one embodiment, the heterologous challenge strain is HSV-2(2.12). Inanother embodiment, the heterologous challenge strain is any other HSV-2strain known in the art.

In one embodiment, the heterologous challenge strain is a different HSVspecies. In another embodiment, in the case of a mutant HSV-1 vaccinestrain, the vaccine confers protection against HSV-2 challenge. Inanother embodiment, in the case of a mutant HSV-2 vaccine strain, thevaccine confers protection against HSV-1 challenge.

In one embodiment, a vaccine as described in the methods andcompositions of the present invention protects a subject against achallenge with a large inoculum of HSV. In another embodiment, the largeinoculum is 10⁶ plaque-forming units (pfu). In another embodiment, theinoculum is 1.5×10⁶ pfu. In another embodiment, the inoculum is 2×10⁶pfu. In another embodiment, the inoculum is 3×10⁶ pfu. In anotherembodiment, the inoculum is 4×10⁶ pfu. In another embodiment, theinoculum is 5×10⁶ pfu. In another embodiment, the inoculum is 7×10⁶ pfu.In another embodiment, the inoculum is 1×10⁷ pfu. In another embodiment,the inoculum is 1.5×10⁷ pfu. In another embodiment, the inoculum is2×10⁷ pfu. In another embodiment, the inoculum is 3×10⁷ pfu. In anotherembodiment, the inoculum is 4×10⁷ pfu. In another embodiment, theinoculum is 5×10⁷ pfu. In another embodiment, the inoculum is 7×10⁷ pfu.In another embodiment, the inoculum is 10⁸ pfu. In another embodiment,the inoculum is 10³-10⁶ pfu. In another embodiment, the inoculum is10³-10⁵ pfu. In another embodiment, the inoculum is 10⁴-10⁶ pfu. Inanother embodiment, the inoculum is 3×10⁴-3×10⁶ pfu. In anotherembodiment, the inoculum is 10⁴-10⁷ pfu. In another embodiment, theinoculum is 3×10⁴-3×10⁷ pfu. In another embodiment, the inoculum is10⁵-10⁸ pfu. In another embodiment, the inoculum is 3×10⁵-3×10⁸ pfu. Inanother embodiment, the inoculum is more than 10⁸ pfu.

In one embodiment, a vaccine as described in the methods andcompositions of the present invention exhibits enhanced safety relativeto gE-containing HSV vaccine strains, due to its inability to infect theganglia. In another embodiment, a method of the present inventionexhibits enhanced safety relative to gE-containing HSV vaccine strains,due to its inability to spread in neurons. In another embodiment, thevaccine provided herein is safe when administered through multipleroutes as exemplified herein (see Example 24).

Various embodiments of dosage ranges of mutant HSV particles can beused, in another embodiment, in methods of the present invention. Inanother embodiment, the dosage is 10³ pfu. In another embodiment, thedosage is 2×10³ pfu. In another embodiment, the dosage is 3×10³ pfu. Inanother embodiment, the dosage is 5×10³ pfu. In another embodiment, thedosage is 10⁴ pfu. In another embodiment, the dosage is 1.5×10⁴ pfu. Inanother embodiment, the dosage is 10⁴ pfu. In another embodiment, thedosage is 2×10⁴ pfu. In another embodiment, the dosage is 3×10⁴ pfu. Inanother embodiment, the dosage is 5×10⁴ pfu. In another embodiment, thedosage is 7×10⁴ pfu. In another embodiment, the dosage is 10⁵ pfu. Inanother embodiment, the dosage is 2×10⁵ pfu. In another embodiment, thedosage is 3×10⁵ pfu. In another embodiment, the dosage is 5×10⁵ pfu. Inanother embodiment, the dosage is 7×10⁵ pfu. In another embodiment, thedosage is 10⁶ pfu. In another embodiment, the dosage is 2×10⁶ pfu. Inanother embodiment, the dosage is 3×10⁶ pfu. In another embodiment, thedosage is 5×10⁶ pfu. In another embodiment, the dosage is 7×10⁶ pfu. Inanother embodiment, the dosage is 10⁷ pfu. In another embodiment, thedosage is 2×10⁷ pfu. In another embodiment, the dosage is 3×10⁷ pfu. Inanother embodiment, the dosage is 5×10⁷ pfu. In another embodiment, thedosage is 7×10⁷ pfu. In another embodiment, the dosage is 10⁸ pfu. Inanother embodiment, the dosage is 2×10⁸ pfu. In another embodiment, thedosage is 3×10⁸ pfu. In another embodiment, the dosage is 5×10⁸ pfu. Inanother embodiment, the dosage is 7×10⁸ pfu.

In another embodiment, the dosage is 10³ pfu/dose. In anotherembodiment, the dosage is 2×10³ pfu dose. In another embodiment, thedosage is 3×10³ pfu/dose. In another embodiment, the dosage is 5×10³pfu/dose. In another embodiment, the dosage is 10⁴ pfu/dose. In anotherembodiment, the dosage is 1.5×10⁴ pfu/dose. In another embodiment, thedosage is 10⁴ pfu/dose. In another embodiment, the dosage is 2×10⁴pfu/dose. In another embodiment, the dosage is 3×10⁴ pfu/dose. Inanother embodiment, the dosage is 5×10⁴ pfu/dose. In another embodiment,the dosage is 7×10⁴ pfu/dose. In another embodiment, the dosage is 10⁵pfu/dose. In another embodiment, the dosage is 2×10⁵ pfu/dose. Inanother embodiment, the dosage is 3×10⁵ pfu/dose. In another embodiment,the dosage is 5×10⁵ pfu/dose. In another embodiment, the dosage is 7×10⁵pfu/dose. In another embodiment, the dosage is 10⁶ pfu/dose. In anotherembodiment, the dosage is 2×10⁶ pfu/dose. In another embodiment, thedosage is 3×10⁶ pfu/dose. In another embodiment, the dosage is 5×10⁶pfu/dose. In another embodiment, the dosage is 7×10⁶ pfu/dose. Inanother embodiment, the dosage is 10⁷ pfu/dose. In another embodiment,the dosage is 2×10⁷ pfu/dose. In another embodiment, the dosage is 3×10⁷pfu/dose. In another embodiment, the dosage is 5×10⁷ pfu/dose. Inanother embodiment, the dosage is 7×10⁷ pfu/dose. In another embodiment,the dosage is 10⁸ pfu/dose. In another embodiment, the dosage is 2×10⁸pfu/dose. In another embodiment, the dosage is 3×10⁸ pfu/dose. Inanother embodiment, the dosage is 5×10⁸ pfu/dose. In another embodiment,the dosage is 7×10⁸ pfu/dose. In another embodiment, the dose is morethan 10⁸ pfu. In another embodiment, the dose is 10³-10⁶ pfu. In anotherembodiment, the dose is 10³-10⁵ pfu. In another embodiment, the dose is10⁴-10⁶ pfu. In another embodiment, the dose is 3×10⁴-3×10⁶ pfu. Inanother embodiment, the dose is 10⁴-10⁷ pfu. In another embodiment, thedose is 3×10⁴-3×10⁷ pfu. In another embodiment, the dose is 10⁵-10⁸ pfu.In another embodiment, the dose is 3×10⁵-3×10⁸ pfu.

In another embodiment, the dose of mutant HSV particles administered toa subject is the above-described dose per gram body weight. In oneembodiment, the dose of mutant HSV particles administered to a subjectis 2.5×10⁴ pfu/gram body weight.

In one embodiment, the methods of the present invention compriseadministering to a subject or contacting a subject with a mutant HSV ofthe present invention and with a herpes simplex virus subunit vaccine,which in one embodiment, is described in WO 2008/085486, published 17Jul. 2008, which is incorporated by reference herein in its entirety. Inone embodiment, a mutant HSV of the present invention and a subunitvaccine are administered at one time, while in another embodiment, amutant HSV is administered and then, at a later time point, a subunitvaccine is administered, while in another embodiment, a subunit vaccineis administered and then, at a later time point, a mutant HSV isadministered.

In one embodiment, the time period separating a first and secondadministration of a mutant HSV, or of a mutant HSV and another vaccinecomposition is 3-6 weeks. In another embodiment, the first and secondadministration (or contacting) are 1 week apart. In another embodiment,the first and second administration (or contacting) are 2 weeks apart.In another embodiment, the first and second administration (orcontacting) are 3 weeks apart. In another embodiment, the first andsecond administration (or contacting) are 4 weeks apart. In anotherembodiment, the first and second administration (or contacting) are 5weeks apart. In another embodiment, the first and second administration(or contacting) are 6 weeks apart. In another embodiment, the first andsecond administration (or contacting) are 7 weeks apart. In anotherembodiment, the first and second administration (or contacting) are 8weeks apart. In another embodiment, the first and second administration(or contacting) are 1 month apart. In another embodiment, the first andsecond administration (or contacting) are 2 months apart.

It is to be understood that in one embodiment, methods of the presentinvention described hereinabove as comprising the step of contacting asubject with a mutant HSV strain, wherein the mutant strain contains aninactivating mutation in a Us8 gene encoding a gE protein may furthercomprise the step of a second contacting (or administration) of saidsubject with the same or another mutant HSV strain.

In one embodiment, “treating” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedhereinabove. Therefore, in one embodiment, compositions for use in themethods of the present invention are administered to/contacted with asubject before exposure to HSV. In another embodiment, compositions foruse in the methods of the present invention are administeredto/contacted with a subject after exposure to HSV.

Thus, in one embodiment, treating may include directly affecting orcuring, suppressing, inhibiting, preventing, reducing an incidence,reducing the severity of, delaying the onset of, reducing symptomsassociated with the disease, disorder or condition, or a combinationthereof. Thus, in one embodiment, “treating” refers inter alia todelaying progression, expediting remission, inducing remission,augmenting remission, speeding recovery, increasing efficacy of ordecreasing resistance to alternative therapeutics, or a combinationthereof. In another embodiment, treating refers to reducing thepathogenesis of, ameliorating the symptoms of, ameliorating thesecondary symptoms of, or prolonging the latency to a relapse of aHerpes Simplex Virus (HSV) infection in a subject. In one embodiment,“preventing” refers, inter alia, to delaying the onset of symptoms,preventing relapse to a disease, decreasing the number or frequency ofrelapse episodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

In one embodiment, symptoms are primary, while in another embodiment,symptoms are secondary. In one embodiment, “primary” refers to a symptomthat is a direct result of the subject viral infection, while in oneembodiment, “secondary” refers to a symptom that is derived from orconsequent to a primary cause. In one embodiment, the compositions andstrains for use in the present invention treat primary or secondarysymptoms or secondary complications related to HSV infection.

In another embodiment, “symptoms” may be any manifestation of a HSVinfection, comprising blisters, ulcerations, or lesions on the urethra,cervix, upper thigh, and/or anus in women and on the penis, urethra,scrotum, upper thigh, and anus in men, inflammation, swelling, fever,flu-like symptoms, sore mouth, sore throat, pharyngitis, pain, blisterson tongue, mouth or lips, ulcers, cold sores, neck pain, enlarged lymphnodes, reddening, bleeding, itching, dysuria, headache, muscle pain,etc., or a combination thereof.

The gE protein as described in the methods and compositions of thepresent invention has, in one embodiment, the sequence:

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRLPPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIAHDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDASPQHS GLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEPTHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLS VWACMTCWRRRAWRAVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPSTNGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW (SEQ ID No: 2). Inanother embodiment, the gE protein is a homologue of SEQ ID No: 2. Inanother embodiment, the gE protein is a variant of SEQ ID No: 2. Inanother embodiment, the gE protein is an isomer of SEQ ID No: 2. Inanother embodiment, the gE protein is a fragment of SEQ ID No: 2. Inanother embodiment, the gE protein comprises SEQ ID No: 2.

In another embodiment, the gE protein is encoded by a nucleotidesequence having the sequence:

atggatcgcggggcggtggtggggtttcttctcggtgtttgtgttgtatcgtgcttggcgggaacgcccaaaacgtcctggagacgggtgagtgtcggcgaggacgatcgagcaccagctccggggcctacggggcgcggcccgacccagaaactactatgggccgtggaacccctggatgggtgcggccccttacacccgtcgtgggtctcgctgatgccccccaagcaggtgcccgagacggtcgtggatgcggcgtgcatgcgcgctccggtcccgctggcgatggcgtacgcccccccggccccatctgcgaccgggggtctacgaacggacttcgtgtggcaggagcgcgcggccgtggttaaccggagtctggttattcacggggtccgagagacggacagcggcctgtataccctgtccgtgggcgacataaaggacccggctcgccaagtggcctcggtggtcctggtggtgcaaccggccccagttccgaccccacccccgaccccagccgattacgacgaggatgacaatgacgagggcgaggacgaaagtctcgccggcactcccgccagcgggaccccccggctcccgcctccccccgcccccccgaggtcaggcccagcgcccccgaagtctcacatgtgcgtggggtgaccgtgcgtatggagactccggaagctatcctgattcccccggggagacgacagcacgaacgtctccatccatgccatcgcccacgacgaccagacctactccatggacgtcgtctggttgaggttcgacgtgccgacctcgtgtgccgagatgcgaatatacgaatcgtgtctgtatcacccgcagctcccagaatgtctgtccccggccgacgcgccgtgcgccgcgagtacgtggacgtctcgcctggccgtccgcagctacgcggggtgaccagaacaaaccccccaccgcgctgttcggccgaggctcacatggagcccgtcccggggctggcgtggcaggcggcctccgtcaatctggagttccgggacgcgtccccacaacactccggcctgtatctgtgtgtggtgtacgtcaacgaccatattcacgcctggggccacattaccatcagcaccgcggcgcagtaccggaacgcggtggtggaacagcccctcccacagcgcggcgcggataggccgagcccacccacccgcacgtcggggcccctccccacgcgcccccaacccacggcgccctgcggttaggggcggtgatgggggccgccctgctgctgtctgcactggggttgtcggtgtgggcgtgtatgacctgttggcgcaggcgtgcctggcgggcggttaaaagcagggcctcgggtaaggggcccacgtacattcgcgtggccgacagcgagctgtacgcggactggagctcggacagcgagggagaacgcgaccaggtcccgtggctggcccccccggagagacccgactctccctccaccaatggatccggctttgagatcttatcaccaacggctccgtctgtatacccccgtagcgatgggcatcaatctcgccgccagctcacaacctaggatccggaaggcccgatcgccgttactcccaggcctccgattcgtccgtcttctggtaa (SEQ ID No: 3). In another embodiment, the gE protein isencoded by a nucleotide molecule that a homologue of SEQ ID No: 3. Inanother embodiment, the nucleotide molecule is a variant of SEQ ID No:3. In another embodiment, the nucleotide molecule is an isomer of SEQ IDNo: 3. In another embodiment, the nucleotide molecule is a fragment ofSEQ ID No: 3. In another embodiment, the nucleotide molecule comprisesSEQ ID No: 3.

In another embodiment, the gE protein as described in the methods andcompositions of the present invention has the sequence:

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWRAVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPSTNGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW (SEQ ID No: 4). Inanother embodiment, the gE protein is a homologue of SEQ ID No: 4. Inanother embodiment, the gE protein is a variant of SEQ ID No: 4. Inanother embodiment, the gE protein is an isomer of SEQ ID No: 4. Inanother embodiment, the gE protein is a fragment of SEQ ID No: 4. Inanother embodiment, the gE protein comprises SEQ ID No: 4.

In another embodiment, the gE protein is encoded by a nucleotidesequence having the sequence:

atggatcgcggggcggtggtggggtttcttctcggtgtttgtgttgtatcgtgcttggcgggaacgcccaaaacgtcctggagacgggtgagtgtcggcgaggacgtttcgttgctaccagctccggggcctacggggcgcggcccgacccagaaactactatgggccgtggaacccctggatgggtgcggccccttacacccgtcgtgggtctcgctgatgccccccaagcaggtacccgagacggtcgtggatgcggcgtgcatgcgcgctccggtcccgctggcgatggcatacgcccccccggccccatctgcgaccgggggtctacggacggacttcgtgtggcaggagcgcgcggccgtggttaaccggagtctggttatttacggggtccgagagacggacagcggcctgtataccctgtctgtgggcgacataaaggacccggctcgccaagtggcctcggtggtcctggtggtgcaaccggccccagttccgactccacccccgaccccagccgattacgacgaggatgacaatgacgagggcgagggcgaggacgaaagtctagccggcactcccgccagcgggaccccccggctcccgccttcccccgcccccccgaggtcttggcccagcgcccccgaagtctcacacgtgcgtggggtgaccgtgcgtatggagactccggaagctatcctgttttcccccggggaggcgtttagcacgaacgtctccatccatgccatcgcccacgacgaccagacctacaccatggacgtcgtctggttgaggttcgacgtgccgacctcgtgtgccgagatgcgaatatacgaatcgtgtctgtatcatccgcagctcccagagtgtctgtccccggccgacgctccgtgcgccgcgagtacgtggacgtctcgcctggccgtccgcagctacgcggggtgttccagaacaaaccccccgccgcgctgttcggccgaggctcacatggagcccttcccggggctggcgtggcaggcggcctcagtcaatctggagttccgggacgcgtccccacaacactccgggctgtatctgtgcgtggtgtacgtcaacgaccatattcacgcatggggccacattaccatcaacaccgcggcgcagtaccggaacgcggtggtggaacagcccctcccacagcgcggcgcggatttggccgagcccacccacccgcacgtcggggcccctccccacgcgcccccaacccacggcgccctgcggttaggggcggtgatgggggccgccctgctgctgtctgcgctggggttgtcggtgtgggcgtgtatgacctgttggcgcaggcgtgcctggcgggcggttaaaagcagggcctcgggtaaggggcccacgtacattcgcgtggccgacagcgagctgtacgcggactggagctcggacagcgagggagaacgcgaccaggtcccgtggctggcccccccggagagacccgactctccctccaccaatggatccggctttgagatcttatcaccaacggctccgtctgtatacccccgtagcgatgggcatcaatctcgccgccagctcacaacctttggatccggaaggcccgatcgccgttactcccaggcctccg attcgtccgtcttctggtaa(SEQ ID No: 5). In another embodiment, the gE protein is encoded by anucleotide molecule that a homologue of SEQ ID No: 5. In anotherembodiment, the nucleotide molecule is a variant of SEQ ID No: 5. Inanother embodiment, the nucleotide molecule is an isomer of SEQ ID No:5. In another embodiment, the nucleotide molecule is a fragment of SEQID No: 5. In another embodiment, the nucleotide molecule comprises SEQID No: 5.

In another embodiment, the gE protein as described in the methods andcompositions of the present invention has the sequence:

MARGAGLVFFVGVWVVSCLAAAPRTSWKRVTSGEDVVLLPAPAERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVTNARRSAFPPQPPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETLEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCADMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRAPHPAPSARGPLRLGAVLGAALLLAALGLSAWACMTCWRRRSWRAVKSRASATGPTYIRVADSELYADWSSDSEGERDGSLWQDPPERPDSPSTNGSGFEILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLW (SEQ ID No: 6; this proteinwas mutated in Examples 1-5 herein). In another embodiment, the gEprotein is a homologue of SEQ ID No: 6. In another embodiment, the gEprotein is a variant of SEQ ID No: 6. In another embodiment, the gEprotein is an isomer of SEQ ID No: 6. In another embodiment, the gEprotein is a fragment of SEQ ID No: 6. In another embodiment, the gEprotein comprises SEQ ID No: 6.

In another embodiment, the gE protein is encoded by a nucleotidesequence having the sequence:

atggctcgcggggccgggttggtgttttttgttggagtttgggtcgtatcgtgcctggcggcagcacccagaacgtcctggaaacgggtaacctcgggcgaggacgtggtgttgcttccggcgcccgcggaacgcacccgggcccacaaactactgtgggccgcggaacccctggatgcctgcggtcccctgcgcccgtcgtgggtggcgctgtggcccccccgacgggtgctcgagacggtcgtggatgcggcgtgcatgcgcgccccggaaccgctcgccatagcatacagtcccccgttccccgcgggcgacgagggactgtattcggagttggcgtggcgcgatcgcgtagccgtggtcaacgagagtctggtcatctacggggccctggagacggacagcggtctgtacaccctgtccgtggtcggcctaagcgacgaggcgcgccaagtggcgtcggtggttctggtcgtggagcccgcccctgtgccgaccccgacccccgacgactacgacgaagaagacgacgcgggcgtgacgaacgcacgccggtcagcgttccccccccaaccccccccccgtcgtccccccgtcgcccccccgacgcaccctcgtgttatccccgaggtgtcccacgtgcgcggggtaacggtccatatggagaccctggaggccattctgtttgcccccggggagacgtttgggacgaacgtctccatccacgccattgcccacgacgacggtccgtacgccatggacgtcgtctggatgcggtttgacgtgccgtcctcgtgcgccgatatgcggatctacgaagcttgtctgtatcacccgcagcttccagagtgtctatctccggccgacgcgccgtgcgccgtaagttcctgggcgtaccgcctggcggtccgcagctacgccggctgttccaggactacgcccccgccgcgatgttttgccgaggctcgcatggaaccggtcccggggttggcgtggctggcctccaccgtcaatctggaattccagcacgcctccccccagcacgccggcctctacctgtgcgtggtgtacgtggacgatcatatccacgcctggggccacatgaccatcagcaccgcggcgcagtaccggaacgcggtggtggaacagcacctcccccagcgccagcccgagcccgtcgagcccacccgcccgcacgtgagagccccccatcccgcgccctccgcgcgcggcccgctgcgcctcggggcggtgctgggggcggccctgttgctggccgccctcgggctgtccgcgtgggcgtgcatgacctgctggcgcaggcgctcctggcgggcggttaaaagccgggcctcggcgacgggccccacttacattcgcgtggcggacagcgagctgtacgcggactggagttcggacagcgagggggagcgcgacgggtccctgtggcaggaccctccggagagacccgactctccctccacaaatggatccggctttgagatcttatcaccaacggctccgtctgtatacccccatagcgaggggcgtaaatctcgccgcccgctcaccacctttggttcgggaagcccgggccgtcgtcactcccaggcctcctatccgtccgtcctctggtaa(SEQ ID No: 7; this gene was mutated in Examples 1-5 herein. In anotherembodiment, the gE protein is encoded by a nucleotide molecule that ahomologue of SEQ ID No: 7. In another embodiment, the nucleotidemolecule is a variant of SEQ ID No: 7. In another embodiment, thenucleotide molecule is an isomer of SEQ ID No: 7. In another embodiment,the nucleotide molecule is a fragment of SEQ ID No: 7. In anotherembodiment, the nucleotide molecule comprises SEQ ID No: 7.

In one embodiment, the gE protein is encoded by one of the followingGenBank Accession Numbers: DQ889502, NC_001806, NC_001798, Z86099,X14112, L00036, X02138, and X04798, and any of AJ626469-AJ626498. Inanother embodiment, the gE protein is homologous to a sequence disclosedin one of the above GenBank Accession Numbers. In another embodiment,the gE protein is a variant of a sequence disclosed in one of the aboveGenBank Accession Numbers. In another embodiment, the gE protein is afragment of a sequence disclosed in one of the above GenBank AccessionNumbers.

In one embodiment, a gE protein HSV-1 glycoprotein E (gE) is a virionsurface protein which is necessary for spread in neurons, and in oneembodiment, is necessary for spread along axons in either direction,both to (“retrograde”), and from (“anterograde”), the neuronal cellbody. In another embodiment, gE also facilitates evasion of the hostimmune system by sequestering host antibodies against HSV-1, renderingthem inactive. In one embodiment, a gE-deleted HSV-1 replicates in theskin, but cannot spread along neurons to establish latency or escape thehost's antibody response. Thus, in one embodiment, infection with thelive attenuated gE-deleted HSV-1 or HSV-2 will elicit a robust immuneresponse in the skin and protect the host from future encounters withthe wild-type virus.

In one embodiment, the gE protein is a HSV-1 gE protein. In anotherembodiment, the gE protein is a HSV-1(NS) gE protein. In anotherembodiment, the gE protein is a HSV-1(17) gE protein. In anotherembodiment, the gE protein is a HSV-1(F) gE protein. In anotherembodiment, the gE protein is a HSV-1(KOS) gE protein. In anotherembodiment, the gE protein is a HSV-1(CL101) gE protein. In anotherembodiment, the gE protein is a HSV-1(MacIntyre) gE protein. In anotherembodiment, the gE protein is a HSV-1(MP) gE protein. In anotherembodiment, the gE protein is a HSV-1(17+ syn) gE protein. In anotherembodiment, the gE protein is a HSV-1(HF) gE protein. In anotherembodiment, the gE protein is any other HSV-1 gE protein known in theart.

In one embodiment, the gE protein is a HSV-2 gE protein. In anotherembodiment, the gE protein is a HSV-2(HG52) gE protein. In anotherembodiment, the gE protein is a HSV-2(2.12) gE protein. In anotherembodiment, the gE protein is a HSV-2(MS) gE protein. In anotherembodiment, the gE protein is a HSV-2(186) gE protein. In anotherembodiment, the gE protein is a HSV-2(G) gE protein. In anotherembodiment, the gE protein is any other HSV-2 gE protein known in theart.

In another embodiment, the gE protein is any other HSV-1 or HSV-2 gEprotein which in one embodiment has greater than 80% homology, inanother embodiment, greater than 85% homology, in another embodimentgreater than 95% homology, and in another embodiment greater than 98%homology to one of the gE proteins or nucleic acid sequences listedhereinabove. In another embodiment, the gE protein has 98.6% homology toHSV(NS) or HSV(17), or both.

In one embodiment, the gE protein is any other gE protein known in theart. In another embodiment, the gE protein is encoded by any Us8nucleotide known in the art. In one embodiment, the Us8 gene has anucleic acid sequence that corresponds to that set forth in GenbankAccession Nos: GeneID:2703448 or GeneID:1487360, or encodes a proteinsequence of glycoprotein E, which in one embodiment, corresponds to thatset forth in Genbank Accession Nos: NP_044670.1 or NP_044538.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the Us6 gene. In one embodiment,the Us6 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703444, GeneID:1487358,NC_001806, NC_001798, EU029158, EF177451, EF177450, EF157322, EF157321,EF157320, EF157319, Z86099, AJ004801, X14112, AF147806, AY779754,AY779753, AY779752, AY779751, AY779750, AY517492, AY155225, AB016432,AF021342, U12183, U12182, U12181, U12180, or InterPro:IPR002896, orencodes a protein sequence of glycoprotein D, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044668.1,NP_044536.1, CAA38245, AAB59754, AAA19629, AAA19631, AAA19630, AAK93950,ABS84899, ABM66848, ABM66847, AAW23134, AAW23133, AAW23132, AAW23131,AAW23130, AAS01730, ABM52981, ABM52980, ABM52979, ABM52978, AAN74642,AAO26211, AAL90884, AAL90883, AAK19597, AAA45785, BAA00020, AAB60555,AAB60554, AAB60553, AAB60552, AAA98962, AAA98963, AAA45842, AAA45786,VGBEDZ, CAB06713, CAA32283, AAB72102, or CAB06713.1.

In another embodiment, the gD protein is any other HSV-1 or HSV-2 gDprotein which in one embodiment has greater than 80% homology, inanother embodiment, greater than 85% homology, in another embodimentgreater than 95% homology, and in another embodiment greater than 98%homology to one of the gD proteins or nucleic acid sequences listedhereinabove.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the Us9 gene. In one embodiment,the Us9 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703452 or GeneID:1487362, orencodes a protein sequence of Us9 membrane protein, which in oneembodiment, corresponds to that set forth in Genbank Accession Nos:NP_044672.1 or NP_044540.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the Us5 gene. In one embodiment,the Us5 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703406 or GeneID:1487357, orencodes a protein sequence of glycoprotein J, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044667.1 orNP_044535.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the Us4 gene. In one embodiment,the Us4 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703404 or GeneID:1487356, orencodes a protein sequence of glycoprotein G, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044666.1 orNP_044534.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL53 gene. In one embodiment,the UL53 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703425 or GeneID:1487342, orencodes a protein sequence of glycoprotein K, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044656.1 orNP_044524.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL10 gene. In one embodiment,the UL10 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703379 or GeneID:1487293, orencodes a protein sequence of glycoprotein M, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044611.1 orNP_044479.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL1 gene. In one embodiment,the UL1 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703393 or GeneID:1487292, orencodes a protein sequence of glycoprotein L, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044602.1 orNP_044470.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL22 gene. In one embodiment,the UL22 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703373 or GeneID:1487306, orencodes a protein sequence of glycoprotein H, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044623.1 orNP_044491.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL27 gene. In one embodiment,the UL27 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703455 or GeneID:1487312, orencodes a protein sequence of glycoprotein B, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044629.1 orNP_044497.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL44 gene. In one embodiment,the UL44 gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703410 or GeneID:1487331, orencodes a protein sequence of glycoprotein C, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044646.1 orNP_044514.1.

In one embodiment, the HSV strain of and for use in the methods of thepresent invention comprise an additional inactivating mutation, which inone embodiment, is an inactivation of the UL49a gene. In one embodiment,the UL49a gene has a nucleic acid sequence that corresponds to that setforth in Genbank Accession Nos: GeneID:2703419 or GeneID:1487337, orencodes a protein sequence of glycoprotein N, which in one embodiment,corresponds to that set forth in Genbank Accession Nos: NP_044652.1 orNP_044520.1.

In another embodiment, the additional mutation is in an HSV-1 or HSV-2glycoprotein that, in one embodiment, has greater than 80% homology, inanother embodiment, greater than 85% homology, in another embodimentgreater than 95% homology, and in another embodiment greater than 98%homology to one or more of the glycoproteins listed hereinabove.

In one embodiment, HSV strains of and for use in the instant inventionmay comprise an inactivating mutation in a gene encoding gD, which inone embodiment is Us6. In another embodiment, HSV strains of and for usein the instant invention may comprise an inactivating mutation in a geneencoding gE, which in one embodiment is Us8. In another embodiment, HSVstrains of and for use in the instant invention may comprise aninactivating mutation in a gene encoding gE and in a gene encoding gD.In one embodiment, the Us6 mutation is introduced to attenuate an HSVstrain comprising a Us8 mutation that is highly virulent. In oneembodiment, the Us6 mutation reduces virus entry. Us6 mutations, as wellas any of the mutations of the present invention may be in either HSV-1or HSV-2 or both. In one embodiment, HSV-1 gD and HSV-2 gD have a largedegree of homology. In one embodiment, the amino acid sequences of HSV-1gD and HSV-2 gD have 81% homology, or in another embodiment, greaterthan 80% homology, or in another embodiment, greater than 85% homology,or in another embodiment, greater than 90% homology, or in anotherembodiment, greater than 95% homology. In one embodiment, the nucleicacid sequences of HSV-1 gD and HSV-2 gD have 85% homology, or in anotherembodiment, greater than 80% homology, or in another embodiment, greaterthan 85% homology, or in another embodiment, greater than 90% homology,or in another embodiment, greater than 95% homology.

In one embodiment, the gD protein derived of the methods andcompositions of the present invention has the sequence:

MGGTAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPL (SEQ ID No: 14). Inanother embodiment, the gD protein is a homologue of SEQ ID No: 14. Inanother embodiment, the gD protein is a variant of SEQ ID No: 14. Inanother embodiment, the gD protein is an isomer of SEQ ID No: 14. Inanother embodiment, the gD protein is a fragment of SEQ ID No: 14. Inanother embodiment, the gD protein comprises SEQ ID No: 14. In oneembodiment, the gD amino acid sequence is an HSV-1 amino acid sequence.

In another embodiment, the gD protein is encoded by a nucleotidesequence having the sequence:

gtggccccggcccccaacaaaaatcacggtagcccggccgtgtgacactatcgtccataccgaccacaccgacgaacccctaagggggaggggccattttacgaggaggaggggtataacaaagtctgtctttaaaaagcaggggttagggagttgttcggtcataagcttcagcgcgaacgaccaactaccccgatcatcagttatccttaaggtctcttttgtgtggtgcgttccggtatgggggggactgccgccaggttgggggccgtgattttgtttgtcgtcatagtgggcctccatggggtccgcggcaaatatgccttggcggatgcctctctcaagatggccgaccccaatcgcMcgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagatcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccgtcctcgcaccagcccttgttttactagatacccccccttaatgggtgcgggggggtcaggtctgcggggttgggatgggaccttaactccatataaagcgagtctggaaggggggaaaggcggacagtcgataagtcggtagcgggggacgcgcacctgttccgcctgtcgcacccacagctttttcgcgaaccgtcccgttttcgggat (SEQ ID No: 15). Inanother embodiment, the gD protein is encoded by a nucleotide moleculethat a homologue of SEQ ID No: 15. In another embodiment, the nucleotidemolecule is a variant of SEQ ID No: 15. In another embodiment, thenucleotide molecule is an isomer of SEQ ID No: 15. In anotherembodiment, the nucleotide molecule is a fragment of SEQ ID No: 15. Inanother embodiment, the nucleotide molecule comprises SEQ ID No: 15. Inone embodiment, the gD nucleotide sequence is an HSV-1 nucleotidesequence.

In one embodiment, the gD protein as described in the methods andcompositions of the present invention has the sequence:

MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (SEQ ID No: 16). Inanother embodiment, the gD protein is a homologue of SEQ ID No: 16. Inanother embodiment, the gD protein is a variant of SEQ ID No: 16. Inanother embodiment, the gD protein is an isomer of SEQ ID No: 16. Inanother embodiment, the gD protein is a fragment of SEQ ID No: 16. Inanother embodiment, the gD protein comprises SEQ ID No: 16. In oneembodiment, the gD amino acid sequence is an HSV-2 amino acid sequence.

In another embodiment, the gD protein is encoded by a nucleotidesequence having the sequence:

atggggcgtt tgacctccgg cgtcgggacg gcggccctgc tagttgtcgc ggtgggactccgcgtcgtct gcgccaaata cgccttagca gacccctcgc ttaagatggc cgatcccaatcgatttcgcg ggaagaacct tccggttttg gaccagctga ccgacccccc cggggtgaagcgtgtttacc acattcagcc gagcctggag gacccgttcc agccccccag catcccgatcactgtgtact acgcagtgct ggaacgtgcc tgccgcagcg tgctcctaca tgccccatcggaggcccccc agatcgtgcg cggggcttcg gacgaggccc gaaagcacac gtacaacctgaccatcgcct ggtatcgcat gggagacaat tgcgctatcc ccatcacggt tatggaatacaccgagtgcc cctacaacaa gtcgttgggg gtctgcccca tccgaacgca gccccgctggagctactatg acagctttag cgccgtcagc gaggataacc tgggattcct gatgcacgcccccgccttcg agaccgcggg tacgtacctg cggctagtga agataaacga ctggacggagatcacacaat ttatcctgga gcaccgggcc cgcgcctcct gcaagtacgc tctccccctgcgcatccccc cggcagcgtg cctcacctcg aaggcctacc aacagggcgt gacggtcgacagcatcggga tgctaccccg ctttatcccc gaaaaccagc gcaccgtcgc cctatacagcttaaaaatcg ccgggtggca cggccccaag cccccgtaca ccagcaccct gctgccgccggagctgtccg acaccaccaa cgccacgcaa cccgaactcg ttccggaaga ccccgaggactcggccctct tagaggatcc cgccgggacg gtgtcttcgc agatcccccc aaactggcacatcccgtcga tccaggacgt cgcgccgcac cacgcccccg ccgcccccag caacccgggcctgatcatcg gcgcgctggc cggcagtacc ctggcggtgc tggtcatcgg cggtattgcgttttgggtac gccgccgcgc tcagatggcc cccaagcgcc tacgtctccc ccacatccgggatgacgacg cgcccccctc gcaccagcca ttgttttact ag (SEQ ID No: 17). Inanother embodiment, the gD protein is encoded by a nucleotide moleculethat a homologue of SEQ ID No: 17. In another embodiment, the nucleotidemolecule is a variant of SEQ ID No: 17. In another embodiment, thenucleotide molecule is an isomer of SEQ ID No: 17. In anotherembodiment, the nucleotide molecule is a fragment of SEQ ID No: 17. Inanother embodiment, the nucleotide molecule comprises SEQ ID No: 17. Inone embodiment, the gD nucleic acid sequence is an HSV-2 nucleic acidsequence.

In one embodiment, an inactivating mutation in a gene encoding gDcomprises a mutation in which an alanine at amino acid 3 of HSV-1 gD orHSV-2 gD is mutated to a cysteine (A3C). In another embodiment, aninactivating mutation in a gene encoding gD comprises a mutation inwhich an alanine at residue 3 of HSV-1 gD or HSV-2 gD is mutated to acysteine (A3C), a tyrosine at residue 2 to alanine (Y2A), a leucine atresidue 4 to alanine (L4A), or a combination thereof. In anotherembodiment, an inactivating mutation in a gene encoding gD comprises adeletion of the alanine at residue 3 of HSV-1 gD or HSV-2 gD, a deletionof the tyrosine at residue 2, a deletion of leucine at residue 4, or acombination thereof. In another embodiment, an inactivating mutation ina gene encoding gD comprises a mutation at amino acid positions 38, 222,223, 215, or a combination thereof. In another embodiment, aninactivating mutation in a gene encoding gD comprises a Y38C mutation,while in another embodiment, it comprises a R222N, F223I, D215Gmutation, or combination thereof.

In another embodiment, an inactivating mutation in a gene encoding gDcomprises mutations in amino acids 2 and 3, 3 and 4, 2-4, 1-5, 1-7, or1-10. In another embodiment, an inactivating mutation in a gene encodinggD consists essentially of a mutation in which an alanine at amino acid3 of HSV-1 gD or HSV-2 gD is mutated to a cysteine (A3C). In anotherembodiment, an inactivating mutation in a gene encoding gD consists of amutation in which an alanine at amino acid 3 of HSV-1 gD or HSV-2 gD ismutated to a cysteine (A3C). In one embodiment, the numbering used todescribe the location of the mutation refers to amino acid numbering ofthe mature peptide after cleaving of the signal sequence, which in oneembodiment, is the first 25 amino acids for HSV-1 or HSV-2 gD, as isknown in the art.

As provided herein and in one embodiment, a mutant HSV strain of thepresent invention comprising a mutation in gD has reduced virulence(Example 16) and ability to reach DRG (Example 17). In anotherembodiment, vaccination with a mutant HSV strain comprising a mutationin gD of the present invention protects against latent HSV infection(Example 18) after subsequent infection with virulent HSV. In anotherembodiment, the vaccination protects against disease caused by orassociated with latent HSV infection. In another embodiment, thevaccination does not itself cause significant disease.

In one embodiment, the composition comprising a mutant HSV strain of thepresent invention further comprises an adjuvant. In one embodiment, theadjuvant comprises a CpG oligonucleotide. In another embodiment, theadjuvant comprises an aluminum salt. In another embodiment, the adjuvantcomprises both a CpG oligonucleotide and an aluminum salt. In anotherembodiment, the adjuvant comprises any other adjuvant disclosedhereinabove. In another embodiment, the adjuvant comprises anycombination of adjuvants disclosed hereinabove. An appropriate dose ofadjuvant may readily be titrated by a skilled artisan and is routine inthe art.

In another embodiment, the booster vaccination follows a single primingvaccination. “Priming vaccination” refers, in another embodiment, to avaccination that comprises a mutant HSV of the present invention. Inanother embodiment, the term refers to a vaccine initially administered.

In another embodiment, a single booster vaccination is administeredafter the priming vaccination. In another embodiment, two boostervaccinations are administered after the priming vaccination. In anotherembodiment, three booster vaccinations are administered after thepriming vaccination.

In one embodiment, the priming and booster vaccinations of the presentinvention are administered at a single site, while in anotherembodiment, they are administered at separate sites.

In some embodiments, any of the mutant HSV strains of and for use in themethods of this invention will comprise an inactivating mutation of thepresent invention, in any form or embodiment as described herein. Insome embodiments, any of the mutant HSV strains of this invention willconsist of an inactivating mutation of the present invention, in anyform or embodiment as described herein. In some embodiments, the mutantHSV strains of this invention will consist essentially of aninactivating mutation of the present invention, in any form orembodiment as described herein. In some embodiments, the term “comprise”refers to the inclusion of the inactivating mutation, such as a mutationin gE or in gD, as well as inclusion of other mutations that may beknown in the art. In some embodiments, the term “consisting essentiallyof” refers to a strain, whose only functional mutation is the indicatedfunctional mutation, however, other mutations may be included that arenot involved directly in the utility of the strain. In some embodiments,the term “consisting” refers to a strain, which contains mutation of aparticular gene or a particular mutation.

In one embodiment, plasmid complementation may be used to complement theinactivating mutation, which in one embodiment, allows at least oneround of infection with a mutant HSV of the invention.

In one embodiment, the present invention provides a composition forimpeding formation of zosteriform lesions in a subject, the compositioncomprising a mutant HSV strain of the present invention.

In one embodiment, the present invention provides a composition forimpeding herpetic ocular disease in a subject, the compositioncomprising a mutant HSV strain of the present invention.

In one embodiment, the present invention provides a composition forvaccinating a subject against an HSV infection, the compositioncomprising a mutant HSV strain of the present invention.

In one embodiment, the present invention provides a composition forimpeding HSV infection in a subject, the composition comprising a mutantHSV strain of the present invention.

In one embodiment, the present invention provides a composition forimpeding herpes-mediated encephalitis in a subject, the compositioncomprising a mutant HSV strain of the present invention.

In one embodiment, a gE protein of the present invention is homologousto a peptide disclosed or enumerated herein. The terms “homology,”“homologous,” etc., when in reference to any protein or peptide, refer,in one embodiment, to a percentage of amino acid (AA) residues in thecandidate sequence that are identical with the residues of acorresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

Homology is, in one embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. For example,computer algorithm analysis of nucleic acid sequence homology caninclude the utilization of any number of software packages available,such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST EnhancedAlignment Utility), GENPEPT and TREMBL packages.

In one embodiment, “homology” refers to identity to a sequence selectedfrom SEQ ID No: 2-7 of greater than 70%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No: 2-7of greater than 72%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 2-7 of greater than 75%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 2-7 of greater than 78%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 2-7 of greater than 80%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:2-7 of greater than 82%. In another embodiment, “homology” refers toidentity to a sequence selected from SEQ ID No: 2-7 of greater than 83%.In another embodiment, “homology” refers to identity to one of SEQ IDNo: 2-7 of greater than 85%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 2-7 of greater than 87%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 2-7 of greater than 88%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 2-7 of greater than 90%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:2-7 of greater than 92%. In another embodiment, “homology” refers toidentity to a sequence selected from SEQ ID No: 2-7 of greater than 93%.In another embodiment, “homology” refers to identity to one of SEQ IDNo: 2-7 of greater than 95%. In another embodiment, “homology” refers toidentity to a sequence selected from SEQ ID No: 2-7 of greater than 96%.In another embodiment, “homology” refers to identity to one of SEQ IDNo: 2-7 of greater than 97%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 2-7 of greater than 98%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 2-7 ofgreater than 99%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 2-7 of 100%.

In one embodiment, homology is determined via determination of candidatesequence hybridization, methods of which are well described in the art(See, for example, “Nucleic Acid Hybridization” Hames B D and Higgins SJ, Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y). In other embodiments, methods of hybridizationare carried out under moderate to stringent conditions, to thecomplement of a DNA encoding a native caspase peptide. Hybridizationconditions being, for example, overnight incubation at 42° C. in asolution comprising: 10-20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA.

Protein and/or peptide homology for any AA sequence listed herein isdetermined, in one embodiment, by methods well described in the art,including immunoblot analysis, or via computer algorithm analysis of AAsequences, utilizing any of a number of software packages available, viaestablished methods. Some of these packages include the FASTA, BLAST,MPsrch or Scanps packages, and, in another embodiment, employ the use ofthe Smith and Waterman algorithms, and/or global/local or BLOCKSalignments for analysis, for example.

In one embodiment, “variant” refers to an amino acid or nucleic acidsequence (or in other embodiments, an organism or tissue) that isdifferent from the majority of the population but is still sufficientlysimilar to the common mode to be considered to be one of them, forexample splice variants.

In one embodiment, “isomer” refers to one of any of two or moresubstances that are composed of the same elements in the sameproportions but differ in chemical and/or bological properties becauseof differences in the arrangement of atoms, which in one embodiment arestereoisomers, in another embodiment, constitutional isomers ortautomers. In one embodiment, an isomer is an optical isomer orentantiomer, a geometric isomer, a D- and L-isomer, positional isomer,or a cis-trans isomer.

In one embodiment of the present invention, “nucleic acids” or“nucleotide” refers to a string of at least two base-sugar-phosphatecombinations. The term includes, in one embodiment, DNA and RNA.“Nucleotides” refers, in one embodiment, to the monomeric units ofnucleic acid polymers. RNA is, in one embodiment, in the form of a tRNA(transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA(messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA(miRNA) and ribozymes. The use of siRNA and miRNA has been described(Caudy A A et al., Genes & Devel 16: 2491-96 and references citedtherein). DNA can be, in other embodiments, in form of plasmid DNA,viral DNA, linear DNA, or chromosomal DNA or derivatives of thesegroups. In addition, these forms of DNA and RNA can be single-, double-,triple-, or quadruple-stranded. The term also includes, in anotherembodiment, artificial nucleic acids that contain other types ofbackbones but the same bases. In one embodiment, the artificial nucleicacid is a PNA (peptide nucleic acid). PNA contain peptide backbones andnucleotide bases and are able to bind, in one embodiment, to both DNAand RNA molecules. In another embodiment, the nucleotide isoxetane-modified. In another embodiment, the nucleotide is modified byreplacement of one or more phosphodiester bonds with a phosphorothioatebond. In another embodiment, the artificial nucleic acid contains anyother variant of the phosphate backbone of native nucleic acids known inthe art. The use of phosphothiorate nucleic acids and PNA are known tothose skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353-57; and Raz N K et al. Biochem BiophysRes Commun 297:1075-84. The production and use of nucleic acids is knownto those skilled in art and is described, for example, in MolecularCloning, (2001), Sambrook and Russell, Eds., and Methods in Enzymology:Methods for molecular cloning in eukaryotic cells (2003) Purchio and G.C. Fareed.

In one embodiment, the present invention provides a kit comprising acompound or composition utilized in performing a method of the presentinvention. In another embodiment, the present invention provides a kitcomprising a composition, tool, or instrument of the present invention.

“Contacting,” in one embodiment, refers to directly contacting thetarget cell with a mutant HSV strain of the present invention. Inanother embodiment, “contacting” refers to indirectly contacting thetarget cell with a mutant HSV strain of the present invention. Thus, inone embodiment, methods of the present invention include methods inwhich the subject is contacted with a mutant HSV strain which is broughtin contact with the target cell by diffusion or any other activetransport or passive transport process known in the art by whichcompounds circulate within the body.

In one embodiment of the methods of the present invention, the mutantHSV strain is carried in the subjects' bloodstream to the target cell.In another embodiment, the mutant HSV strain is carried by diffusion tothe target cell. In another embodiment, the mutant HSV strain is carriedby active transport to the target cell. In another embodiment, themutant HSV strain is administered to the subject in such a way that itdirectly contacts the target cell.

Pharmaceutical Compositions and Methods of Administration

In one embodiment, the methods of the present invention compriseadministering a pharmaceutical composition comprising the mutant HSVstrain and a pharmaceutically acceptable carrier.

“Pharmaceutical composition” refers, in one embodiment, to atherapeutically effective amount of the active ingredient, i.e. themutant HSV strain, together with a pharmaceutically acceptable carrieror diluent. A “therapeutically effective amount” refers, in oneembodiment, to that amount which provides a therapeutic effect for agiven condition and administration regimen.

The pharmaceutical compositions containing the mutant HSV strain can be,in one embodiment, administered to a subject by any method known to aperson skilled in the art, such as parenterally, transmucosally,transdermally, intramuscularly, intravenously, intra-dermally,subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially,intra-vaginally or intra-tumorally.

In another embodiment as described in the methods and compositions ofthe present invention, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e. as a solid or a liquid preparation. Suitable solidoral formulations include tablets, capsules, pills, granules, pelletsand the like. Suitable liquid oral formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment of the present invention, the active ingredient is formulatedin a capsule. In accordance with this embodiment, the compositions ofthe present invention comprise, in addition to the active compound andthe inert carrier or diluent, a hard gelating capsule.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intra-muscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intra-arterially and are thus formulated in a formsuitable for intra-arterial administration. In another embodiment, thepharmaceutical compositions are administered intra-muscularly and arethus formulated in a form suitable for intra-muscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the mutant HSV strain is prepared and applied as asolution, suspension, or emulsion in a physiologically acceptablediluent with or without a pharmaceutical carrier.

In another embodiment, the pharmaceutical compositions provided hereinare controlled-release compositions, i.e. compositions in which themutant HSV strain is released over a period of time afteradministration. Controlled- or sustained-release compositions includeformulation in lipophilic depots (e.g. fatty acids, waxes, oils). Inanother embodiment, the composition is an immediate-release composition,i.e. a composition in which all the mutant HSV strain is releasedimmediately after administration.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXPERIMENTAL DETAILS SECTION Example 1 HSV_((gE null)) does not CauseDisease Materials and Experimental Methods (Examples 1-4)

Virus Strains

Wild-type HSV-1 strain NS, a low-passage-number clinical isolate, wasused for generation of mutant viruses. To construct HSV-1_((gE null)),the entire gE coding sequence was excised from pCMV3gE-1 with XbaI andcloned into pSPT18. pSPT18 has the sequence:

(SEQ ID No: 1) gaatacaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattccggtctccctatagtgagtcgtattaatttcgataagccagctgggcctcgcgcgatcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcaccgatcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtaccccctggaagctccctcgtgcgctctcctgaccgaccctgccgcttaccggatacctgtccgcctactccatcgggaagcgtggcgattctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatatcacctagatccattaaattaaaaatgaagattaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagagcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgagccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgatactgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagagctatgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgacttcggggcgaaaactctcaaggatcttaccgctgagagatccagttcgatgtaacccactcgtgcacccaactgatcacagcatatttactacaccagcgtactgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggaccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccatcgtctcgcgcgatcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgaggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacgggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatctggctagcgatgaccctgctgattggttcgctgaccatttccgggtgcgggacggcgttaccagaaactcagaaggttcgtccaaccaaaccgactctgacggcagatacgagagagatgatagggtctgcttcagtaagccagatgctacacaattaggcttgtacatattgtcgttagaacgcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata.

A 1.1-kb HpaI-BglII fragment from amino acids (AA) 124-508 was excised,and the HpaI site was changed to a BglII site. A 4.3-kb fragment derivedfrom pD6P containing the Escherichia coli β-galactosidase gene (β-gal)under the control of the HSV ICP6 promoter was cloned into the BglIIsite. The resultant vector contains 374 bp of NS DNA sequences 5′ and225 bp 3′ of the ICP6::lacZ cassette and was used to construct the gEnull virus. The XbaI fragment containing the flanking sequence vectorwas isolated, and 750 ng was cotransfected into Vero cells with 1.0 μgof NS DNA by calcium phosphate transfection. The DNA-calcium phosphatemixture was removed, and cells were shocked with 15% glycerol. Cellswere harvested when cytopathic effects were noted in 30-40% of cells andwere sonicated to prepare a virus pool. Recombinant gE null virusexpressing β-gal was selected by infecting Vero cells and overlayingwith 0.5% agarose, 5.0% fetal bovine serum (FBS), and 300 μg of5-bromo-D-galactopyranoside (X-Gal). Blue plaques were picked andpurified twice in X-Gal agarose overlay and once by limiting dilution.Virus was purified from supernatant fluids of infected Vero cells on a5-70% sucrose gradient.

HSV-1_((Rescue gE null)) was prepared by co-transfection of Vero cellswith 1.0 μg of NS-gEnull DNA and 1.5 μg of wild-type gE fragmentpurified from pCMV3gE-1, which was obtained by digesting HSV-1 (NS) DNAwith NruI. Progeny viruses were examined by immunoperoxidase stainingusing anti-gE MAb 1BA10 to confirm expression of gE on the surface ofinfected cells. Plaques were purified by limiting dilution, and viruspools were prepared.

Virus stocks were grown on confluent Vero cells (an African green monkeykidney epithelial cell line) at an MOI of 2.0. 24 hours post-infection,cells were scraped in the media and centrifuged at 3,000×g. All but 1 mLof supernatant was removed, and cells were resuspended, sonicated for 3seconds and distributed into 50 mcL (microliter) aliquots. For mockinfections, similar aliquots were made using uninfected cells.

Mouse Flank Infection Protocol

All experimental protocols were approved by the University ofPennsylvania animal and laboratory resources IACUC committee. Five- tosix-week-old BalbC mice (National Cancer Institute) were allowed toacclimate to the biosafety level 2 animal facility with constanttemperature and photoperiod (12 hours of light, 12 hours of darkness)for 1 week. Mice were shaved and depilated with depilatory cream (Nair™)along the right flank (for vaccination) or the left flank (forchallenge), then washed with warm water. The next day, mice wereanesthetized via intraperitoneal injection of 75 mcL of 14.3 mg/mlketamine and 1.8 mg/ml xylazine in PBS, then infected by making 60superficial scratches in a 1 cm² area of the flank, 1 cm dorsal to thespine, with a 26⅜-gauge needle through a 10 mcL droplet containing5×10³-5×10⁵ pfu HSV (FIG. 1). In HSV flank-infected mice, secondaryspread back to the skin from the neurons of one or more spinal gangliaresults in a belt-like lesion (FIG. 2). Mice were sacrificed byasphyxiation with CO₂, followed by cervical dislocation. Mice wereobserved at 24-hour intervals starting at day 3 post-inoculation torecord the appearance and severity of skin lesions and illness. Astandardized scoring system to describe lesion severity was used toprovide consistency in observations (FIG. 3).

Vaccination and Challenge

For vaccination, mice were infected by making scratches through theinoculum, containing either HSV-1(gE null) or virus-free cell lysate(described above), on the right flank. Mice were challenged 28 dayslater on the opposite (left) flank by scratching through a dropletcontaining 1×10⁵ pfu HSV-1(NS).

Dissections of Dorsal Root Ganglia and Skin

Dorsal root ganglia (DRG) along either the right (for analysis ofHSV-1_((gE null)) vaccination or HSV-1_((Rescue gE null)) infection), orleft (for analysis of HSV-1(NS) challenge) sides of the spinal columnwere removed, pooled, and placed in 110 mcL DMEM (2.5% FBS) and frozenat −80° C. until analysis.

A 1-cm² area of skin at the site of inoculation was removed. Half of thesample was placed in a tube containing 110 mcL DMEM (2.5% FBS) andfrozen at −80° C. until analysis. The other half was placed on an indexcard with like-samples and immersed in 4% paraformaldehyde in 1×PBS for24 hours at 4° C., then the solution was replaced with 1×PBS. Samplesremained at 4° C. until processing for histological analysis.

Sectioning, Histology and Immunohistochemistry

Paraffin embedding, sectioning and staining of skin samples wasperformed by the Pathology Core Facility at Children's Hospital ofPhiladelphia. Skin sections were stained for HSV-1 antigen usinganti-HSV-1 rabbit polyclonal antibody (DAKO) and counter-stained withhematoxylin.

Results

Groups of five mice were flank-infected by scarification of 10^(3.5),10^(4.5), or 10^(5.5) plaque-forming units (pfu) of the vaccine strain,HSV-1_((gE null)). For comparison to the virulent form of HSV-1, anothergroup of five mice was flank-infected with 10⁵⁵ pfu ofHSV-1_((Rescue gE null)), the vaccine strain backbone with the geneencoding gE restored. HSV-1_((Rescue gE null)) infection resulted inillness and 60% mortality (FIG. 4). However, no clinical signs ofdisease, illness or death occurred following HSV-1_((gE null))vaccination.

In addition, the HSV-1_((Rescue gE null)) infection caused severeprimary lesions that appeared ulcerative and necrotic (FIG. 5). Incontrast, HSV-1_((gE null)) infection caused a mild skin pathology atthe site of inoculation indistinguishable from that of mock vaccination.Thus, all or essentially all of the skin pathology following theadministration of HSV-1_((gE null)) resulted from the process of scratchinoculation itself.

These findings show that infection with gE null herpes viruses does notcause disease.

Example 2 HSV_((gE null)) does not Spread within Sensory Neurons

HSV-1_((Rescue gE null)) infection caused severe secondary (zosteriform)ulcerative and necrotic lesions that first appeared at 4-5 (dayspost-infection) dpi (FIG. 6). In contrast, no secondary lesions wereseen following HSV-1_((gE null)) infection. Since secondary lesionformation along the dermatome depends on the ability of the virus tospread along neurons from the skin to the ganglia, and then back again,these results show that the vaccine is not able to spread within sensoryneurons. Therefore, HSV_((gE null)) is unable to cause recurrentinfection.

Example 3 HSV_((gE null)) Replicates within Skin Cells

To determine the extent of replication of HSV-1_((gE null)) in skincells, mice (n=3) were infected with HSV-1_((gE null)) or virulentHSV-1_((Rescue gE null)). On days 1, 3, 6, 8 and 13 post-inoculation,mice were sacrificed, skin at the site of inoculation was removed, andDRG from the right side of each mouse were pooled. Titering to determineviral content of the homogenized tissue revealed that HSV-1_((gE null))replicated in skin following vaccination, but less extensively thanHSV-1_((Rescue gE-null)) (FIG. 7). Further, HSV-1_((gE null))-vaccinatedskin was infiltrated by immune cells, and the virus was cleared by day 6(FIG. 8).

These results show that HSV_((gE null)) replicates in the skin, and thusis expected to elicit an inflammatory response by the host.

Levels of HSV-1_((Rescue gE null)) exceeded that of HSV-1_((gE null)) by4 orders of magnitude (FIG. 9). In addition, no infectious virus wasdetected in DRG of mice infected with HSV-1_((gE null)). The absence ofthe characteristic belt-like pattern of lesions and of detectable virusin the ganglia confirmed that no neuronal viral spread occurred afterHSV-1_((gE null)) vaccination.

Thus, HSV infection in the absence of gE is limited to the skin of thehost, and the immune system is able to detect and clear the virusrapidly.

Example 4 HSV-1_((gE null)) Vaccination is Protective Against Wild-TypeHSV-1 Infection

Mock-vaccinated or HSV-1_((gE null)) vaccinated mice were challengedwith a lethal dose of 10⁵ pfu WT HSV-1 (NS strain) 28 days aftervaccination. Whereas 100% of mock-vaccinated mice died followingchallenge, 100% of HSV-1_((gE null))-vaccinated mice survived the lethalchallenge (FIG. 10). All tested doses of HSV-1_((gE null)) (10^(3.5),10^(4.5), and 10^(5.5) pfu) were protective against challenge.Additionally, very little primary (inoculation site) disease wasobserved after challenge of the HSV-1_((gE null))-vaccinated mice (FIG.11). Vaccinated mice had undetectable levels of challenge virus in skin,at least 5 orders of magnitude less than mock-vaccinated mice (FIG. 12).

In contrast to the severely diseased mock-vaccinated mice, primarydisease healed rapidly in vaccinated mice. Confirming this observation,immunohistochemistry of equivalent skin samples demonstrated thepresence of very little antigen in vaccinated mice on day 3, andclearance by day 6 (FIG. 13). Histological analyses also revealed thatvaccinated mice had significant infiltration of immune cells, showingthat the vaccine successfully primed the host immune system.

Unlike the severe dermatome lesions resulting from zosteriform spread ofthe virus in mock-vaccinated mice, no zosteriform disease was seen invaccinated mice (FIG. 14). In addition, viral titers in pulverized DRGwere undetectable in vaccinated mice and thus, at least 4 orders ofmagnitude less than in mock-vaccinated mice (FIG. 15).

Example 5 HSV_((gE null)) Vaccination Prevents Establishment of LatentWild-Type HSV-1 Infection after Subsequent Challenge Materials andExperimental Methods

Recovery of Infectious Virus from Tissues

DRG and skin samples were removed from mice immediately after sacrifice(at 1, 3, 6, 8 or 13 dpi). Three mice were infected for each data point,but like tissues from these mice were analyzed individually. Tissueswere placed in 110 mcL DMEM containing 2.5% FBS and stored at −80° C.until analysis. To determine viral titer, tissue was thawed andpulverized with a disposable pestle. Infectious virus in 50 mcL of thesupernatant, serially diluted (1:10, 1:100 and 1:1000), was quantifiedby plaque assay on confluent Vero cells in 12-well dishes.

Explant of DRG

To recover infectious virus from latently infected mice, DRG wereremoved from the same (right) side of infection with HSV-1_((gE null)).All DRG from an individual mouse were placed in one well of a 12-welldish of confluent Vero cells bathed in DMEM (2.5% FBS). Medium waschanged every 2 days and cells were monitored for signs of CPE, anindication that DRG contained infectious virus.

Results

DRG were removed from vaccinated mice, 28 days post-challenge, andexplanted in order to reactivate latent challenge virus. Reactivationfrom associated neurons of only 1/15 of the vaccinated mice occurredupon removal (FIG. 16).

Results of this and the previous Example show that HSV_((gE null))protects mice from neuronal infection upon challenge and from developinglatent infection typically associated with WT HSV infections.

Example 6 Vaccination of Mice with HSV-1 gE Cross Protects Against HSV-2Challenge Materials and Experimental Methods

Female Balb/C mice, 6-8 weeks old, were acclimated to the animalfacility for 10 days. Mice were anesthetized and shaved and hair wasremoved by Nair treatment of the right flank. The following day,anesthetized mice were mock-vaccinated or vaccinated with 5×10⁵ pfuHSV-1ΔgE (which is referred to, in one embodiment, as gE null) byscratching 60 times through a 10 mcl (microliter) droplet of inoculumwith a 26 (⅝)-gauge needle. The opposite flank (left side) of each mousewas shaved and denuded as before, twenty-seven days later. Mice werechallenged the following day (day 28) by scratch inoculation of 10⁵ pfuHSV-2 (strain 2.12). Mice were observed and scored daily for inoculationsite disease, zosteriform disease and survival. (Scoring: 0=no disease4=severe necrotic disease). Error bars represent the standard error ofthe mean (SEM).

Results

To test the ability of HSV-1ΔgE vaccination to protect against HSV-2disease, HSV-1ΔgE-vaccinated mice were epidermally challenged withwild-type HSV-2. None of the HSV-1ΔgE vaccinated mice died, while 80% ofmock vaccinated mice died (FIG. 17, top panel). Vaccinated miceexhibited inoculation site disease that healed rapidly compared to theunvaccinated mice (FIG. 17, middle panel). Additionally, vaccinated micewere completely protected from the zosteriform disease and deathobserved in the mock-vaccinated mice (FIG. 17, bottom panel).

Thus, ΔgE HSV vaccination is capable of protecting subjects againstheterologous HSV disease, even of a different species of herpes simplex.

Example 7 Vaccination with HSV-1ΔgE Protects Against HSV-1(KOS) andInhibits Establishment of HSV-1 Latency Materials and ExperimentalMethods

Vaccination and assessment of disease were performed as described forthe previous Example, except that 5×10⁵ pfu HSV-1, strain KOS, was usedfor the challenge. For measurement of latent infection, mice weresacrificed 41 d post-challenge, and DRG from both right and left sideswere removed, placed in DMEM/10% FBS, minced with scissors, andexplanted onto sub-confluent Vero cell monolayers. Cultures weremonitored daily for 20 d for plaque formation, indicative ofreactivation from latency.

Results

This experiment tested the ability of HSV-1ΔgE vaccination to protectmice from the disease associated with a heterologous HSV-1 wild-typestrain. HSV-1ΔgE-vaccinated mice challenged with wild-type HSV-1, strainKOS, exhibited inoculation site disease that healed rapidly compared tounvaccinated mice (FIG. 18, top panel). Additionally, HSV-1ΔgEvaccination protected HSV-1 KOS-challenged mice completely againstzosteriform disease (FIG. 18, middle panel). Since HSV-1 KOS infectionof mice is not lethal, this strain was utilized to compare viralreactivation from latency in mock-versus HSV-1ΔgE-vaccinated mice at 4weeks post-challenge. In mock-vaccinated mice, HSV-1(KOS) virusreactivated from explanted DRG in 100% of mice (n=10), whereas only 1/10HSV-1ΔgE vaccinated mice (10%) exhibited reactivation (FIG. 18, table inbottom panel), which could have been latent infection by either thevaccine or the challenge virus. Therefore, HSV-1ΔgE vaccination iseffective at protecting mice from both disease and establishment oflatency by heterologous HSV viruses.

Example 8 Vaccination with HSV-1ΔgE Protects Against HSV-1 VaginalChallenge

Vaccination was performed as described for Example 6.Medroxyprogesterone acetate (2 mg) (Sicor Pharmaceuticals, Inc., IrvineCalif.), diluted to 100 mcl total volume in a 0.9% NaCl/10 mM HEPESbuffer, was injected subcutaneously 33 days later into the neck area ofeach mouse. Five days later (day 38), mice were anesthetized,intra-vaginally swabbed with a calcium alginate swab dipped in PBS, andchallenged by intra-vaginal instillation of 5×10⁵ pfu HSV-1(strain NS).Mice were allowed to recover in their cages, resting in a proneposition. Challenged mice were observed daily for vaginal disease andsurvival. Daily intra-vaginal swabs were taken for analysis by viraltitering on Vero cells.

Results

The ability of HSV-1ΔgE vaccination to protect against vaginal challengewith wild-type HSV-1 was tested. All mice vaccinated with HSV-1ΔgEsurvived the challenge, whereas 60% of mock-vaccinated mice succumbed(FIG. 19A, top panel). All mock-vaccinated mice showed some sign ofvisible disease in the vaginal area; however HSV-ΔgE-vaccinated miceshowed no obvious visible signs of disease (FIG. 19B). By day 1post-vaginal challenge, HSV-1ΔgE-vaccinated mice had 100-fold lessinfectious virus than mock-vaccinated animals, as detected in vaginalswab samples. By day three post-challenge, no infectious virus wasdetected in swabs from HSV-1ΔgE-vaccinated mice, a value that is atleast 30,000-fold less than mock-vaccinated mice on the same day.Additionally, infection of HSV-1ΔgE-vaccinated mice cleared nearly threetimes faster than mock-vaccinated mice (FIG. 19A, bottom panel). Thus,vaccination with HSV-1ΔgE protects from death and disease associatedwith HSV-1 vaginal challenge and confers the ability to rapidly clearHSV infection.

Example 9 Vaccination with HSV-1ΔgE by Epidermal, Subcutaneous, andIntramuscular Routes Protects Against HSV-1 Challenge Materials andExperimental Methods

Vaccination was performed with 5×10⁵ pfu HSV-1ΔgE by scratching 60 timesthrough a 10 mcl droplet of inoculum with a 26 (⅝)-gauge needle,injection of 100 mcl inoculum into the scruff of the necksubcutaneously, or by intramuscular injection of 100 mcl inoculum intothe right rear thigh muscle.

For measurement of latent infection, mice were sacrificed 32 dayspost-challenge, and DRG from both right and left sides were removed,placed in DMEM/10% FBS, minced with scissors and explanted ontosub-confluent Vero cell monolayers. Cultures were monitored daily (for15 days) for plaque formation, indicating reactivation from latency.

Results

The efficacy of intramuscular and subcutaneous routes of administrationof the HSV-1ΔgE vaccine was compared to epidermal scarification used inprevious Examples. All routes of vaccination were effective atprotecting mice against death upon epidermal challenge with HSV-1(NS)(FIG. 20, top panel). Mice vaccinated by each of the three routesexhibited inoculation site disease upon HSV-1(NS) challenge that wasonly slightly greater than mock-challenged mice (FIG. 20, bottom leftpanel). HSV-1ΔgE vaccination by epidermal scarification or intramuscularinjection protected mice completely against zosteriform disease (FIG.20, bottom right panel). Following challenge, 1/5 mice vaccinated by thesubcutaneous route had several discrete zosteriform lesions, which werenot severe and resolved quickly (FIG. 20, bottom right panel). Inaddition, the ability of the vaccine to prevent latent infection wasmeasured. HSV-1ΔgE vaccination by all routes protected against theestablishment of latency (Table 1). Mock-vaccinated mice showed 100%reactivation (Table 1; also see FIG. 18).

TABLE 1 HSV-1 vaccination by different routes protects against HSVlatency. Vaccination Route Reactivation from Latency Mock 1 of 1Epidermal Scarification 0 of 4 Intra-Muscular 0 of 5 Subcutaneous 1 of 5

Thus, HSV-1ΔgE administered by epidermal, intramuscular or subcutaneousroutes each protects against acute disease, flares and latent disease bywild-type HSV challenge.

Example 10 Vaccination with HSV-1ΔgE by Epidermal Scarification,Subcutaneous Injection and Intramuscular Injection Induces NeutralizingAntibodies Materials and Experimental Methods

Mice were vaccinated as described in the previous Example. On day 21,mice were bled through jugular veins. On day 28, the opposite flank(left side) of each mouse was shaved and denuded as before.Neutralization assays on serum samples were done by incubating 50 mclserum dilution (1:10 to 1:320) with 10² pfu HSV-1(NS) in 5 mcl for 1hour at 37° C., and then inoculating Vero cell monolayers.

Results

The efficacy of HSV-1ΔgE vaccination by the epidermal scarification,intramuscular, and subcutaneous routes of administration for inductionof neutralizing antibodies were measured. HSV-1ΔgE vaccination of miceby all three routes induced neutralizing antibody formation; theepidermal scarification and intramuscular routes yielded significantlyhigher levels than subcutaneous vaccination (FIG. 21).

Example 11 Vaccination with HSV-1ΔgE Protects Against Flank Challengewith Various Heterologous HSV-1 Strains

Ability of HSV-1ΔgE vaccination to protect against heterologous, highervirulence wild-type HSV-1 strains (F and 17) was measured; withvaccination and challenged performed as described for Example 7, exceptthat challenge utilized various strains. HSV-1ΔgE vaccination protectedmice completely from death upon epidermal challenge with HSV-1(NS),HSV-1(F) and HSV-1(17) (FIG. 22, top panel). Vaccination also reducedinoculation site disease, although challenge with HSV-1(F) causedslightly more disease at the inoculation site than HSV-1(NS) orHSV-1(17) (FIG. 22, middle panel). Moreover, HSV-1ΔgE vaccinationcompletely protected all mice challenged with HSV-1(NS) and HSV-1(17)from zosteriform disease and ⅔ mice challenged with HSV-1(F); the othermouse challenged with HSV-1(F) had two small zosteriform lesions (FIG.22, bottom panel). Thus, HSV-1ΔgE vaccination protects mice againstvarious heterologous strains of HSV-1.

Example 12 Vaccination with HSV-1ΔgE Protects Against Flank Challengewith Doses Up to 1×10⁷ Pfu of HSV-1(NS)

The ability of HSV-1ΔgE vaccination to protect against higher doses ofwild-type HSV-1 was measured; with vaccination and challenged performedas described for Example 7, except that challenge utilized higher dosesof 10⁵, 10⁶ or 10⁷ pfu of HSV-1(NS). Vaccinated mice were all completelyprotected from death and zosteriform disease (FIG. 23, top and bottompanels). Vaccinated mice challenged with 10⁵ pfu HSV-1(NS) exhibitedinoculation site disease that was slightly more severe thanmock-challenged mice, indicating that most of the disease was caused bythe scarification (needle scratch). Challenge of HSV-1ΔgE vaccinatedmice with 10⁶ or 10⁷ pfu was associated with significantly reduceddisease at the inoculation site, which healed rapidly compared withmock-vaccinated mice (middle panel).

Example 13 Characterization and Stability of HSV gD Mutant Materials andExperimental Methods (Examples 13-18)

Virus Strains

Wild-type HSV-1 strain KOS was used to prepare gD mutants. To constructHSV-1_((gD null)), plasmid pSC594 was constructed by inserting A3C(alanine to cysteine) and Y38C (tyrosine to cysteine) mutations intoplasmid pRM416 which contains the KOS gD open reading frame flanked by474 base pairs 5′ and 985 base pairs 3′ of the open reading frame. HSV-1gD-mull DNA and pSC594 DNA were co-transfected into VD60 cells.Recombinant virus was screened by replication in Vero cells and thenplaque-purified. After each plaque purification, 600 base pairs wereamplified by PCR at the 5′ end of the gD gene that included the sites ofthe mutations. The amplified gD fragments were screened by restrictionenzyme mapping. Introduction of a new SspI site confirmed the presenceof the A3C mutation and the loss of an RsaI site confirmed the presenceof the Y38C mutation. Following further plaque purification, DNAsequencing was used to confirm the presence of the mutations. The cloneswere grown to high titer on Vero cells, purified on a 10% to 60% sucrosegradient, and subjected to a final DNA sequence analysis and restrictionmapping, which revealed that only the A3C mutation remained. TheKOS-gDA3C was further purified on a sucrose gradient and the entire gDgene was sequenced to confirm the presence of the A3C mutation and theabsence of additional unintended mutations.

Rescued KOS-gDA3C virus, referred to as rKOS-gDA3C, was generated byco-transfection of Vero cells with KOS-gDA3C and pRM416 DNA.

Virus stocks were grown in Dulbecco's minimum essential medium (DMEM),supplemented with 10% fetal calf serum (FCS). B78-H1 cells, mousemelanoma cells that are non-permissive for HSV-1 entry, were grown inDMEM with 5% FCS. B78-H1-A10 cells (A10) and B78-H1-C10 cells (C10)stably express HVEM and nectin-1, respectively, and were grown in DMEMcontaining 5% FCS and 500 μg/ml of G418. The gD-null virus waspropagated in Vero cells stably transfected with gD DNA (VD60 cells).HSV-1 strain NS, a low-passage clinical isolate, was used for challengestudies in mice. Viruses were grown in Vero cells, unless otherwisenoted, purified on sucrose gradients and stored at −80° C.

Mouse Flank Infection Protocol

All experimental protocols were approved by the University ofPennsylvania animal and laboratory resources IACUC committee.Five-six-week-old Balb/c mice (Charles River) were allowed to acclimateto the biosafety level 2 animal facility with constant temperature andphotoperiod (12 hours of light, 12 hours of darkness) for 1 week. Micewere shaved and depilated with depilatory cream (Nair™) along the rightflank (for vaccination) or the left flank (for challenge), then washedwith warm water. The next day, mice were anesthetized viaintraperitoneal injection of 75 mcL of 14.3 mg/ml ketamine and 1.8 mg/mlxylazine in PBS, then infected by making 60 superficial scratches in a 1cm² area of the flank, 1 cm dorsal to the spine, with a 30-gauge needlethrough a 10 mcL droplet containing 5×10⁵ pfu HSV. Mice were observed at24-hour intervals starting at day 3 post-inoculation to record theappearance and severity of skin lesions and illness. Scores at theinoculation site ranged from 0 to 5 and at the zosteriform site from 0to 10. One point was assigned per vesicle or if lesions were confluentmultiple points were assigned based on the size of the confluentlesions.

Entry Assay

KOS-gDA3C, rKOS-gDA3C or KOS (400 pfu) was incubated for one hour at 4°C. with B78-H1, A10, C10 or Vero cells. Cells were warmed to 37° C. for0, 10, 30, 60 or 120 minutes followed by washing to remove unbound virusand exposed to a citrate buffer pH 3.0 wash for 1 minute to inactivatevirus that had bound but had not entered cells. After an additionalwash, cells were overlaid with 0.6% low-melt agar in DMEM, and plaqueswere visualized and counted after 68 hours.

Single-Step and Multi-Step Growth Curves

Single-step growth curves were performed on B78-H1, A10 and C10 cellsinoculated with KOS, KOS-gDA3C or rKOS-gDA3C virus at an MOI of 3. Afterone hour at 37° C., cells were treated with citrate buffer pH 3.0 forone minute, and cells and supernatant fluids were collected immediately(time 0) or at 2, 4, 8, 10, 12, 20 and 24 hours. Samples werefreeze-thawed once, sonicated three times each for 10 seconds andtitered on Vero cells. Multi-step growth curves were performed in asimilar fashion, except infection was performed at an MOI of 0.01 andtiters measured at 24, 48 and 72 hours.

Real-Time Quantitative PCR for Viral DNA in Dorsal Root Ganglia (DRG)

DRG nearest the site of inoculation were harvested and DNA was isolatedusing the Qia Amp-mini DNA kit (Qiagen). The Us9 gene was amplified toquantify viral genome copy number in DRG. The PCR reaction was performedin a 50 mcl volume with a minimum of 200 ng of DNA from DRG. Fifty pmolof forward 5′cgacgccttaataccgactgtt (SEQ ID NO: 8) and reverse5′acagcgcgatccgacatgtc (SEQ ID NO: 9) primers and 15 pmol of Taqmanprobe 5′tcgttggccgcctcgtcttcgct (SEQ ID NO: 10) were added. One unit ofAmpli Taq Gold (Applied Bioscience) per 50 mcl reaction was added. Realtime PCR amplification was performed on an ABI Prism7700 SequenceDetector (Applied Biosystems). A standard curve was generated frompurified HSV-1 (NS) DNA. Mouse adipsin, a cellular housekeeping gene wasalso amplified from DRG DNA under identical conditions. The primers usedfor amplification were forward 5′gatgcagtcgaaggtgtggtta (SEQ ID NO: 11)and reverse 5′cggtaggatgacactcgggtat (SEQ ID NO: 12), while Taqman probe5′tctcgcgtctgtggcaatggc (SEQ ID NO: 13) was used for detection. Theviral DNA copies were then normalized based on the murine adipsin copynumber.

Results

Since the gD transcript is co-terminal 3′ with gI and gJ, the molecularmass of gD and gI was evaluated by western blots of cells infected withWT, rKOS-gDA3C, or KOS-gDA3C virus. The size of the proteins was similarfor the three viruses (FIG. 24A), while DNA sequencing confirmed theintegrity of the gJ gene in KOS-gDA3C (result not shown). The stabilityof the gDA3C mutation was confirmed by restriction digestion using SspIof PCR-amplified DNA fragments to confirm the presence of the cysteineresidue at position 3. The Ssp1 site was maintained through 30 passages,suggesting that the change of alanine to cysteine at residue 3 wasstable (FIG. 24B). This was confirmed by DNA sequence analysis afterevery five passages.

Mice were scratch-inoculated on the flank with KOS-gDA3C, and DRGharvested five days post-infection to confirm the stability of the gDA3Cmutation in vivo. Virus was isolated from three individual plaques. Allthree isolates retained the Ssp1 site (FIG. 24C), suggesting that thecysteine residue at amino acid 3 was maintained, which was confirmed byDNA sequencing.

Example 14 HSV gD Mutant as an Entry-Impaired Live Virus Vaccine

The entry of KOS, rKOS-gDA3C, and KOS-gDA3C into cells that express HVEM(A10), nectin-1 (C10), both (Vero), or neither receptor (B78-H1) wasevaluated. Entry of the three viruses into Vero cells was comparable(FIG. 25A), while each virus failed to enter B78-H1 cells (FIG. 25B).Entry of KOS-gDA3C into A10 cells was reduced by approximately 50%compared with KOS or rKOS-gDA3C (FIG. 25C), and entry into C10 cells wasreduced by approximately 70% (FIG. 25D).

These findings show that the gDA3C mutation reduces entry mediated byboth HVEM and nectin-1 receptors.

Example 15 Growth Curves of HSV gD Mutant Virus

Virus replication was examined by performing single-step growth curvesat an MOI of 3. KOS, rKOS-gDA3C and KOS-gDA3C failed to infect B78-H1cells (results not shown). Replication of the three viruses wascomparable in A10 cells (FIG. 26A) and C10 cells (FIG. 26B), except thatthe titers of KOS-gDA3C were reduced at time 0 (at the end of theone-hour adsorption period), which reflects the entry defect seen inExample 14.

Multi-step growth curves were performed by infecting the cells at an MOIof 0.01 to allow multiple cycles of virus replication. Compared with KOSand rKOS-gDA3C, peak titers of KOS-gDA3C were reduced at 72 hours byapproximately 1.5 log 10 in A10 cells (FIG. 26C) and 2 log 10 in C10cells (FIG. 26D).

Example 16 HSV gD Mutant has Reduced Virulence

The virulence of the KOS-gDA3C mutant was evaluated in the mouse flankmodel. Mice were infected with 5×10⁵ PFU of KOS, rKOS-gDA3C, orKOS-gDA3C and animals scored for disease at the inoculation andzosteriform sites. Mice infected with KOS-gDA3C had less severe diseaseat the inoculation site (FIG. 27A) and almost no zosteriform diseasewith only one of 30 mice developing 3 lesions on day 5 (FIG. 27B).Photographs of the zosteriform site disease are shown on day 10 (FIG.27C).

These findings show that infection with the gD mutant herpes viruscauses minimal disease.

Example 17 HSV gD Mutant has Reduced Ability to Reach DRG

Mice were inoculated with 5×10⁵ PFU of KOS, rKOS-gDA3C, or KOS-gDA3C andat 5 days post-infection, the DRG were harvested to measure viral titers(FIG. 28A) and viral genome copy number (FIG. 28B), which were reducedfor KOS-gDA3C compared with KOS or rKOS-gDA3C.

These findings show that the gD mutant herpes virus is defective inreaching the DRG.

Example 18 HSV gD Mutant as an Attentuated Live Virus Vaccine

Mice were mock-infected or infected with rKOS-gDA3C or KOS-gDA3C andallowed to recover. Although rKOS-gDA3C produced extensive disease, allanimals survived, as did all mice infected with KOS. Thirty days later,mice previously infected with KOS-gDA3C or rKOS-gDA3C were challenged onthe opposite flank with HSV-1 strain NS at 10⁶ PFU (approximately 20LD50). The challenge virus caused extensive disease at the inoculation(FIG. 29A) and zosteriform (FIG. 29B) sites in the mock group. KOS-gDA3Cand rKOS-gDA3C protected against disease at the inoculation site andboth viruses totally prevented zosteriform disease. None of therKOS-gDA3C or KOS-gDA3C infected mice died after the NS strainchallenge, while 100% of the mock-infected mice died (result not shown).

These findings show that KOS-gDA3C provided protection against challengethat was comparable to protection provided by the more virulentrKOS-gDA3C.

The ability of a prior infection with KOS-gDA3C to prevent the WT virusfrom reaching the DRG was evaluated. Mice were mock-infected or infectedin the flank with 5×10⁵ pfu of rKOS-gDA3C or KOS-gDA3C. Thirty dayslater, mice were challenged with 10⁶ pfu of NS on the opposite flank.DRG that innervate the challenge site were harvested five dayspost-challenge. NS viral titers were approximately 6 log 10 in DRG ofmice that were previously mock infected, while no virus was recoveredfrom DRG of mice previously infected with rKOS-gDA3C or KOS-gDA3C (FIG.29C).

Quantitative PCR was performed on the DRG at five days post-challenge.Approximately 5.8 log 10 HSV-1 genome copies were detected in DRG ofpreviously mock-infected mice compared with 3.4 or 3.2 log 10 DNA copiesin mice previously infected with rKOS-gDA3C or KOS-gDA3C, respectively(FIG. 29D).

These findings show that KOS-gDA3C is attenuated in causing skin lesionsat the inoculation and zosteriform sites and in infecting DRG, yet it isas effective as rKOS-gDA3C in protecting mice against WT HSV-1challenge.

These examples suggest that an HSV strain with a mutation in gD may beused as an attenuated live HSV vaccine. FIG. 30 shows a model in whichat each step of the virus life cycle, less KOS-gDA3C is produced becauseof the defect in virus entry. These steps include the amount of virusproduced in epidermal cells (labeled E), in DRG nuclei (labeled N), andthat return to the skin at the zosteriform site.

Example 19 HSV-2_((gE null)) does not Cause Disease Materials andExperimental Methods

Cells and Viruses

Vero cells (ATCC CCL81) are cultured in Dulbecco's modified Eagle'smedium containing heat-inactivated 10% newborn calf serum (LifeTechnologies, Gaithersburg, Md.) plus 50 micrograms (mcg) ofpenicillin/ml, 50 mcg/ml of streptomycin/ml, and 0.15 mcg/ml ofFungizone® (Life Technologies) at 37° C. and 5% CO₂. Clarified stocks ofHSV-2 strains are prepared from infected Vero monolayers and stored at−80° C. until used. Titers of virus are determined by standard plaqueassays.

FIG. 31A demonstrates sequence alignment between gE of HSV-2(HG52) andHSV-1(NS). FIG. 31B demonstrates sequence alignment between gE ofHSV-2(2.12) and HSV-2(HG52). A deletion in base pairs (bp) 369-1479 ofthe 1635 bp HSV-2 (2.12) Us8 gene, encoding HSV-2 gE, was introduced asfollows. Two PCR fragments from HSV-2(2.12), namely a 658 bp fragmentcorresponding to the region 5′ of the intended deletion and a 536 bpfragment 3′ of the intended deletion, were subcloned into thepBluescript SK+ multiple cloning site (MCS). The 5′ flanking region wassubcloned into the KpnI and HindIII sites of the pBluescript SK+MCS, andthe 3′flanking region was subcloned into the PstI and SacI sites of theMCS. This left a short stretch of the MCS between the 5′ and 3′ flankingregions that includes the EcoRI and EcoRV restriction sites and causes aframeshift such that only the first 123 amino acids of gE were expressed(FIG. 31C). The vector was co-transfected into Vero cells withHSV-2(2.12) genomic DNA to allow for homologous recombination. Thevirion DNA purified from resulting plaques was screened by PCR to detectincorporation of the deletion.

Mouse Vaginal Model of HSV-2 Infection

Mice are treated with 2.0 mg of Depo-Provera (Upjohn, Kalamazoo, Mich.)subcutaneously in the scruff of the neck 7 and 1 day prior to viralinoculation to synchronize their estrus cycles and to increase theirsusceptibility to HSV-2 vaginal infection. HSV-2 virus (10⁴ pfu) isinstilled in the vaginal cavity following wet and then dry vaginalswabbing with a calcium alginate swab (Fisher Scientific, Pittsburgh,Pa.). Animals are assessed daily for symptomatic disease (as indicatedby hair loss and erythema near the vagina) through 14 dayspost-inoculation (p.i.). Survival is followed through 21 days p.i. As anadditional indicator of infection, vaginal swabs are collected andtested for viral content on Vero cells.

Results

We previously showed that HSV-1 NS-gEnull is a safe and effectivevaccine in mice owing to the defect in anterograde neuronal transportand deficient cell to cell spread of this strain. In our most recentwork, we have constructed an HSV-2 gE deletion mutant, HSV-2ΔgE(gfp),that deletes a region of gE-2 similar to the region deleted inNS-gEnull. We have shown that, similar to HSV-1 NS-gEnull, HSV-2ΔgE(gfp)is defective in anterograde spread and deficient in cell-to-cell spread.We also demonstrate that this strain causes no disease in mice andserves as an effective vaccine against HSV-2 epidermal and mucosalchallenge of mice, particularly when given in mice as two dosesseparated by approximately three weeks.

gE null HSV-2 virus was constructed from strain HSV-2(2.12), using asimilar strategy as that used for HSV-1 (Example 1). The HSV-2 mutant,HSV-2ΔgE(gfp), was produced by deleting and inserting the gfp2 cassetteunder the control of a CMV promoter. The gfp2 cassette, which allowedfor screening of recombinant viruses by fluorescence, was inserted inthe US8 reading frame just after the bases encoding amino acid 123. Theportion of the US8 gene encoding the 156 C-terminal amino acids of gEremained but was not expressed. The mutation was made in wild-typestrain HSV-2(2.12) (FIG. 32).

The HSV-2ΔgE(gfp) mutant did not express gE but had normal expression ofgD, VP5 and US9 (FIG. 33). In vitro single-step growth kinetics ofHSV-2ΔgE(gfp) in vitro were similar to WT in both epithelial (Verocells) (FIG. 34A) and primary neuronal (superior cervical ganglia fromrat embryos) (FIG. 34B) cell lines. In Vero cells, HSV-2ΔgE(gfp)produced significantly smaller plaques than HSV-2(2.12) (FIG. 34C),indicating that HSV-2ΔgE(gfp) had impaired cell-to-cell spread. Thus,replication of HSV-2ΔgE(gfp) was normal, but cell to cell spread wasimpaired.

Mouse retina were infected with 4×10⁵ PFU HSV-2(2.12) or HSV-2ΔgE(gfp),and immunofluoresence in the retina and optic nerve of infected micewere observed on days 3 and 5 post-infection. Viral antigen was seen inretinas infected with both viruses (FIG. 35A), suggesting that aproductive infection occurred. However, antigen was not seen in theoptic nerve sections following HSV-2ΔgE(gfp) retina infection (FIG.35B), indicating that the virus was defective in anterograde axonaltransport. Therefore, HSV-2ΔgE(gfp) was defective in anterograde spreadin vivo.

Anterograde and retrograde retinorecipient areas of the brain followingHSV-2ΔgE(gfp) retina infection of the mouse were examined. Mouse retinaswere infected with 4×10⁵ PFU HSV-2ΔgE(gfp) or HSV-2(2.12). HSV-2 antigenappeared in the dorsal lateral geniculate nucleus (LGN) (arrowhead) andthe optic tract (arrow) in HSV-2(2.12) infected mice by 5 dpi but not inthe brains of mice infected with HSV-2ΔgE(gfp) at 5 or 8 days postinfection (dpi; FIG. 36A). HSV-2 antigen was present in the dorsal LGN(arrowhead), the ventral LGN (open arrowhead) and the intergeniculateleaflet (IGL) of the LGN (arrow) in the brains of HSV-2(2.12) infectedmice but not in the brains of mice infected with HSV-2ΔgE(gfp) (FIG.36B). HSV-2 antigen was detected in the superior colliculus (SC)(arrowhead) and the oculomotor and Edinger-Westphal nuclei (arrow) ofmice infected with HSV-2(2.12) infected mice but not in the brains ofmice infected with HSV-2ΔgE(gfp) (FIG. 36C). In mouse retinas infectedwith HSV-2(2.12), virus traveled through both anterograde and retrogradeoptic circuits. However, no spread was observed in either direction inthe brains of mice infected with HSV-2ΔgE(gfp), indicating that thevaccine is defective in both anterograde and retrograde directionalspread in vivo.

Next, the safety of HSV-2ΔgE(gfp) in the mouse flank model wasdetermined Mice were infected by scarification on denuded flank skinwith 5×10⁵ pfu HSV-2(2.12) or HSV-2ΔgE(gfp) and monitored daily forsurvival, inoculation site disease and zosteriform disease. In contrastto HSV-2(2.12) flank infection, HSV-2ΔgE(gfp) did not cause any death(FIG. 37A), produced inoculation site disease similar to mock infection(FIG. 37B), and caused no zosteriform disease (FIG. 37C). Therefore,HSV-2ΔgE(gfp) causes no disease or death following mouse flankscarification and does not reactivate from DRG, indicating that thisvaccine is safe in mice.

Explants:

DRG explanted >28 dpi from 5 mice infected with HSV-2ΔgE did notreactivate virus when cultured on Vero cells for 16 days. There was nopositive control, since all mice infected with HSV-2(2.12) died. Theseresults suggest that HSV-2ΔgE(gfp) is impaired in epithelial cell tocell spread and/or epithelial to neuronal cell spread within the host.

HSV-2ΔgE(gfp) is not Detected in the Skin or the DRG Following MouseFlank Scarification, Further Demonstrating the Safety of this Virus as aVaccine.

Mice were infected by scarification on denuded flank skin with 5×10⁵ pfuHSV-2(2.12) or HSV-2ΔgE(gfp). HSV-2(2.12) flank infection yielded virusin skin samples (FIG. 38A) on all days tested and in DRG on days 3, 6and 8 (FIG. 38B). In contrast, tissues titered from mice infected withHSV-2ΔgE(gfp) had no detectable virus.

HSV-2ΔgE(gfp) and HSV-1(NS)-gEnull Infections Cause No Disease or DeathFollowing Vaginal Inoculation and Yield Lower Swab Titers than WT HSVStrains.

Mice were infected intravaginally with 5×10⁵ pfu of HSV-2(2.12) orHSV-2ΔgE(gfp). All mice infected with the wild-type HSV-2 strain (2.12)died by day 8, whereas only 40% of the mice infected with the wild-typeHSV-1 strain (NS) died (FIG. 39A). 100% of mice infected withHSV-2(2.12) develop severe disease, while mice infected with HSV-1(NS)develop more moderate disease (FIG. 39B). All mice infected with eitherHSV-1 NS-gEnull or HSV-2ΔgE(gfp) vaccine strains survived and showed nosigns of disease indicating that these vaccines are non-pathogenic inmice (FIGS. 39A and B). Mice were swabbed intra-vaginally on days 0-3.Titers of all strains increased from days 1 to 2, indicating that bothWT and vaccine strains are likely replicating (FIG. 39C).

Example 20 HSV-2_((gE null)) Vaccination is Protective Against Wild-TypeHSV-2 Infection

HSV-2ΔgE(gfp) Immunization by IM Route Protects Mice Better than SubQInjection from Epidermal Challenge by Flank Scarification.

Mice were vaccinated with HSV-2ΔgE(gfp) or mock-vaccinated, infected IMor subQ with 5×10⁵ pfu (1,736 LD50s) HSV-2(MS), and survival,inoculation site disease, and zosteriform disease were evaluated. While100% of mock vaccinated mice died following flank scarification ondenuded flank skin with 5×10⁵ pfu (1,736 LD50s) HSV-2(MS), all micegiven HSV-2ΔgE(gfp) by the IM route survived (FIG. 40A). However, only 4of 5 vaccinated by the SubQ route survived. The IM route was also moreeffective at preventing both inoculation site (FIG. 40B) and zosteriformdisease (FIG. 40C) than the SubQ route. One of 5 mice vaccinated IMdeveloped severe zosteriform disease but recovered, but the remaining 4vaccinated mice had no zosteriform lesions.

HSV-2ΔgE(gfp) Given IM to Mice Results in Reduced Viral Loads in Skinand DRG Following Challenge by Epidermal Scarification.

Mice were vaccinated IM with 5×10⁵ pfu HSV-2ΔgE(gfp) or mock-vaccinated,infected with 5×10⁵ pfu (1,736 LD50s) HSV-2(MS), and viral titers wereevaluated in skin (FIG. 41A) and DRG (FIG. 41B). Virus could be detectedin both skin and DRG samples from mock vaccinated mice but not from micevaccinated with HSV-2ΔgE(gfp), indicating that vaccination led toreduced viral loads following challenge.

HSV-2ΔgE(gfp) Immunization by IM Route Protects Mice Better than SubQInjection from Mucosal Challenge with 250 Pfu (50 LD50s) of HSV-2(MS) inthe Mouse Vaginal Model.

Mice were vaccinated IM or subQ with HSV-2ΔgE(gfp) or mock-vaccinated,infected intravaginally with 250 pfu (50 LD50s) HSV-2(MS), and survival,inoculation site disease, and virus titer were evaluated. 100% of mockvaccinated mice died whereas all of the mice vaccinated withHSV-2ΔgE(gfp) by the IM route survived (FIG. 42A). In contrast, only 60%of the mice vaccinated by the SubQ route survived the vaginal challenge,indicating that the IM route is more effective at protecting frommucosal challenge. All mice that were mock vaccinated developed severedisease and some of the mice vaccinated by the SubQ route developeddisease (FIG. 42B). However, mice that were vaccinated withHSV-2ΔgE(gfp) by the IM route, developed no visible signs of disease.SubQ vaccination with HSV-2ΔgE(gfp) caused reduced intravaginal viralloads relative to the mock vaccinated group (FIG. 42C). However, IMvaccination was even more effective at reducing the amount of virus inthe vagina following challenge.

Explants:

Sacral DRG explanted >28 dpi from 4 of 5 mice vaccinated IM withHSV-2ΔgE and 5 of 5 SubQ vaccinated mice reactivated virus when culturedon Vero cells for 16 days. This indicates that one dose of the vaccinegiven by the IM route protected ganglia from challenge infection in aminority of cases.

Two Immunizations with HSV-2ΔgE(gfp) are Significantly Better than Onein Protecting Mice from Disease Following Challenge with 5×10⁴ Pfu (10⁴LD50s) of HSV-2(MS) in the Mouse Vaginal Model.

Mice were vaccinated with either one or two doses (three weeks apart) of5×10⁵ pfu HSV-2ΔgE(gfp) or mock-vaccinated, infected intravaginally with5×10⁵ pfu (1,736 LD50s) HSV-2(MS), and survival, inoculation sitedisease, and vaginal virus titer were evaluated. While 100% of the micefrom the mock vaccinated group died, groups vaccinated with either oneor two doses of HSV-2ΔgE(gfp) were completely protected from death (FIG.43A). One vaccination with HSV-2ΔgE(gfp) did not completely protect micefrom disease; however, when given in two doses, mice showed no outwardsigns of disease (FIG. 43B). While one HSV-2ΔgE(gfp) vaccination reducedvaginal titers following challenge relative to wild-type, twovaccinations were significantly better (FIG. 43C). Both vaccinatedgroups had no detectable virus in swabs taken on day 5, whereasmock-vaccinated animals had approximately 10⁴ pfu challenge virus on day5, persisting at high levels (greater than 10⁴ pfu) until day 7, justprior to death. Photos from each mouse taken on day 7 post-inoculationdemonstrate the difference between vaginal disease in each group (FIG.43D).

Example 21 Efficacy of HSV-2_((gE null)) Vaccination Against ExistingHSV-2 Genital Infection in a Guinea Pig Model Materials and ExperimentalMethods

Guinea Pig Model of Genital Herpes

On the day of inoculation, vaginal closure membranes are ruptured with apre-moistened calcium alginate swab. Vaginal vault is swabbed with a drycalcium alginate swab, and 5×10³ pfu of HSV-2 (strain MS) is instilledinto the vaginal vault with a syringe and a 20-gauge plastic catheter.This dose is generally sublethal, while providing infection of nearlyevery inoculated animal. During acute genital infection, animals areevaluated daily through day 14 p.i. for genital skin disease and urinaryretention. Disease is quantified by a skin lesion scoring system rangingfrom 0 (no disease) to 4 (severe disease characterized by large ulcerswith maceration). Daily scoring of each animal proceeds from day 15-60p.i. to establish frequency of external recurrent herpetic lesions.

Viral Shedding Detection

Guinea pigs spontaneously shed HSV-2 from the vaginal cavity even in theabsence of signs of disease. Viral DNA can be detected in 10 to 20% ofthe vaginal swabs from latently infected guinea pigs, allowing for thestudy of viral shedding frequencies and comparisons of the magnitudes.Vaginal cavities are swabbed daily with a calcium alginate-tipped swabfrom days 15-60 p.i. DNA is extracted from each swab sample using theQIAmp® DNA extraction system (Qiagen, Inc, Chatsworth, Calif.),including mock swab blanks as monitors for sample contamination, andsubjected to quantitative PCR for HSV-2 DNA, using primers targeting theDNA polymerase gene. A separate reaction is performed for each of thespecimens to address template quality and quantity, using a second setof primers to amplify the single-copy guinea pig albumin gene. Theresulting 498-bp amplimer is utilized for normalization of DNAconcentration and a more quantitative estimate of the HSV-2 burden ineach specimen. Positive specimens are compared to amplification of aseries of 10-fold serial dilutions of established genomic equivalentsusing MS HSV-2 stocks. Reactions are run in a GeneAmp® PCR System 9600(Perkin-Elmer Corp, Norwalk, Conn.) beginning with a “hot start” at 95°C. for 2 min; then 35 cycles of denaturation at 95° C. for 1 min,annealing for 1 min at 65° C., and 72° C. extension for 1 min 30 s; anda final 7-min extension at 72° C. Amplification products of each sample,positive and negative controls, and the series of known standards aredetected by Southern blotting. HSV-2 burdens are extrapolated from thelinear relationship established from band density of a dilution seriesof known genomic equivalents amplified in parallel to the samples.

Determination of HSV-2 DNA Copy Numbers in Guinea Pig Dorsal RootGanglia.

Sacral dorsal root ganglia (6-8 per animal) are dissected on day 60 p.i.and weighed, viral DNA is extracted by using a QIAamp® DNA minikit(QIAGEN), and real-time PCR is performed. A standard curve isconstructed for each experiment, using purified plasmid containing HSV-2gD gene sequences. Data are normalized to probes specific for guinea piglactalbumin DNA.

Immunization of guinea pigs: 60 days p.i., guinea pigs are immunized ormock immunized once or twice separated by approximately 3 weeks andanimals followed for recurrent lesions by visual inspection of thevaginal orifice and surrounding skin and by daily swabs for HSV-2 DNAdetected by PCR.

Results

The guinea pig model is utilized to evaluate the efficacy of herpessimplex vaccine against recurrent herpetic disease. This model providesa naturally occurring recurrent disease similar to that seen in humanHSV-2 infections, and latently infected guinea pigs shed virus vaginallyat a frequency similar to that observed in humans.

Guinea pigs previously infected by HSV-2 and then vaccinated withHSV-2_((gE null)) are expected to have significantly reduced frequencyof genital lesion compared to mock-vaccinated animals and reduce thenumber of animals that experience any recurrences. In addition,HSV-2_((gE null)) vaccination is expected to significantly reduce themagnitude of viral shedding.

When HSV-2_((gE null)) vaccination is given prior to challenge toevaluate the effect of the vaccination on the establishment of latentHSV-2 infection, accumulation of wt HSV-2 viral genomes in guinea pigDRG is evaluated on day 60 post challenge. HSV-2_((gE null)) vaccinationis expected to significantly reduce the number of viral genomes in theDRG.

This and the previous Example are expected to provide additionalevidence that HSV-2_((gE null)) vaccines are efficacious in protectingsubjects against HSV-2 infection and subsequent genital reactivation.

Example 22 Introduction of Additional Deletions to the Us Region inOrder to Further Impair the Anterograde Spread of the ΔgE-2 VaccineStrain

In order to further attenuate the ΔgE-2 vaccine strain, additionaldeletions are introduced into Us7 and Us9, encoding the gI and Us9proteins, using a similar approach to that used to construct the HSV-2Us8 deletion (FIG. 32). A cloning vector that contains two 500-1000 basepair flanking regions, each homologous to either the DNA sequence 5′ or3′ of the intended deletion, is constructed. The DNA for these tworegions is obtained by PCR of HSV-2(2.12) genomic DNA. The cloningvector is co-transfected with HSV-2 genomic DNA, so that the deletionsare incorporated into the viral DNA by homologous recombination. Theresulting plaques are screened for the correct Us deletion by PCR.

Example 23 Identification of Additional Mutations that ImpairAnterograde Spread of the ΔgE-2 Vaccine Strain

RNAi gene silencing methodology is utilized to identify genes other thangE, Us7 and Us9 that are involved in virus spread. RNAi technology usesapproximately 20-22 base-pair double-stranded RNA fragments withsequences identical to the viral gene targeted for silencing. To targetsequences on viral genes of HSV-1 or HSV-2, small RNA double-strandedfragments identical in sequence to the viral RNA are synthesized usingstandard techniques known in the art, and are introduced by transfectiontechnology into cells that are then infected with HSV-1 or HSV-2wild-type or mutant virus. Spread of defective virus is detected byscreening for small plaques in human epidermal keratinocytes (HaCaT)cells (Collins W J et al. Herpes simplex virus gE/gI expressed inepithelial cells interferes with cell-to-cell spread. J Virol. 2003February; 77(4):2686-95). The genes targeted by the RNAi fragments thatinduce small plaques are used in gene deletion studies. Inactivatingmutations are then introduced into the gene or genes identified by theabove RNAi screening method to create mutant viruses. Spread propertiesof mutant viruses are evaluated in vitro using rat superior cervicalganglion cell neuron cultures (Wang F, Tang W, McGraw H M, Bennett J,Enquist L W, and Friedman H M. J. Virol 79:13362-72, 2005) and the mouseretina eye infection model (Wang F, Tang W, McGraw H M, Bennett J,Enquist L W, Friedman H M. J. Virol 79:13362-72, 2005). The viral mutantstrains identified that modify spread in vitro or in vivo are introducedinto strains containing deletions of gE, Us7 or Us9 to develop strainscontaining deletions in multiple genes to identify the optimumcombination of mutations that causes little or no disease wheninoculated into laboratory animals, that results in low levels or noviral DNA in DRG, and that provides maximum protection against diseaseand establishment of viral latency when challenged by infection withwild type HSV-1 or HSV-2.

In other experiments, efforts are focused on virion membrane proteins,e.g. glycoproteins J, G, K, and M. Membrane glycoproteins required forvirus entry, e.g. glycoproteins B, D, H and L, are excluded. Thesevirion membrane proteins are analyzed as described in the previousparagraph.

Example 24 HSV-2ΔgE(gfp) Vaccines are Safe

The LD₅₀ of the HSV-2ΔgE(gfp) were assessed (labeled as gE2-del virus inthe table) in BALB/c and SCID mice. The LD₅₀ was calculated by theReed-Muench method. After infection, disease and death were monitoredfor 4 weeks. At least 5 mice were evaluated at each inoculation dose.

IM Safety

In BALB/c mice, wild-type virus was evaluated at 5×10³ to 5×10⁵ PFU, andthe vaccine strain at 5×10⁵ to 5×10⁶ PFU. In SCID mice, wild type viruswas used at 5×10′ to 5×10⁵ PFU, while the vaccine strain was evaluatedat 5×10⁴ to 5×10⁶ PFU.

Intravaginal Infection

In BALB/c mice, wild-type virus was used at 1-50 PFU, while the vaccinestrain was evaluated at 5×10⁴ to 5×10⁵ PFU. In SCID mice, wild-typevirus was used at 5×10′ to 5×10³ PFU, while the vaccine strain was usedat 5×10⁴ to 5×10⁶ PFU.

Intravenous Infection:

In BALB/c mice wild-type virus was used at 5×10³ to 5×10⁵ PFU, while thevaccine strain was used at 5×10⁵ to 5×10⁶ PFU. In SCID mice, wild-typevirus was evaluated at 5×10³ to 5×10⁵ PFU, while the vaccine strain wasevaluated at 5×10⁴ to 5×10⁶ PFU.

Intracranial Infection

BALB/c mice were inoculated with wild-type virus at 5 to 5×10³ PFU,while the vaccine strain was inoculated at 5×10⁴ to 5×10⁶ PFU.

Results

No mice died or showed signs of illness at any dose of HSV-2ΔgE(gfp)(gE2-del virus) inoculated by these routes (highest inoculum was 5×10⁶PFU for all routes, except vaginal route in BALB/c mice that was at5×10⁵ PFU) (FIG. 44). In contrast, mice inoculated with HSV-2 strain2.12 (the parental virus for vaccine strain) died at doses that were 10²to 10⁵ PFU lower than the highest dose of vaccine virus that caused nodisease) (FIG. 44). The LD50 of the vaccine virus were also evaluatedafter intracranial inoculation of 3-4 week old BALB/c mice injected with25 μl of virus. 9/10 mice inoculated with 5×10⁶ PFU of the vaccinestrain died, and 1/5 mice died at 5×10⁵ PFU (LD50=1.4×10⁶ PFU). Incontrast, the LD50 of WT virus was <5 PFU, which was the lowest dosetested (FIG. 44). Therefore, there is >2.8×10⁵ PFU difference in theLD50 comparing HSV-2ΔgE(gfp) with wild-type virus following intracranialinoculation.

Example 25 Antibody Response to HSV-2 gD-2 after Immunization withHSV-2ΔgE(gfp)

Mice were bled prior to immunization (prebleed) or immunized with HSV-2glycoprotein gD as a positive control. The gD-2 protein extends fromamino acid 26-331 (amino acid 26 as the first amino acid after thesignal sequence). The gD-2 construct has 306 amino acids and is referredto as bac-gD-2(306t) High-level expression and purification of secretedforms of herpes simplex virus type 1 glycoprotein gD synthesized bybaculovirus-infected insect cells. HSV-2 gD was used at 2 μg/mouse mixedwith CpG and alum as adjuvants, where CpG: TCC ATG ACG TTC CTG ACG TT(SEQ ID NO: 19) 50 μg per mouse was mixed with alum 25 μg/μg protein ina 50 μl volume. Mice were immunized IM in the calf three times separatedby 2 week intervals (labeled gD imm+ct). For comparison, mice wereimmunized IM in the calf muscle with HSV-2ΔgE(gfp) using 5×10⁵ PFU,given either once or twice separated by 4 weeks. Mice were bled 4 weeksafter the first immunization (1×imm), 4 weeks after the secondimmunization (2×imm), and 3 and 5 months after the second immunization(3 mo, or 5 mo). n=5 mice per group, except gD imm (+ct), which involvesa single mouse.

Results

No antibodies to gD-2 were detected in the pre-immune sera. Antibodytiters after gD-2 immunization (positive control) were higher thanantibodies produced to the live virus vaccine HSV-2ΔgE(gfp) whenadministered once or twice (FIG. 45) Importantly, no antibody responseto gD-2 was detected after the first immunization; however, antibodieswere detected after the second immunization that persisted for 3 and 5months (FIG. 45).

Example 26 Antibody Response to HSV-2 gC-2 after Immunization withHSV-2ΔgE(gfp)

A similar experiment was performed as described above for gD-2; however,the mice were immunized with HSV-2 gC (gC-2) as the positive control andantibody was measured to gC-2 protein. The gC-2 immunogen extends fromamino acid 27-426 (amino acid 1 is the methionine at the ATG site, andamino acid 27 is the first amino acid after the signal sequence). Basedon the cloning method, an aspartic acid and proline were added at theamino terminus just prior to amino acid 27. The gC-2 protein is referredto as bac-gC-2(426t). Mice were immunized with gC-2 5 μg mixed with CpGand alum given 3 times separated by 2 weeks. Other mice were immunizedwith HSV-2ΔgE(gfp) at 5×10⁵ PFU in the calf muscle given either once ortwice, separated by 4 weeks. n=5 mice per group, except gC imm (+ct),which involves a single mouse.

Results

No gC-2 antibody was detected in pre-immune serum (labeled Prebleed).High titers of gC-2 antibody were detected following immunization withgC-2 mixed with CpG and alum (labeled as gC imm+ct) (FIG. 46). Lowtiters of gC-2 antibody were detected after one immunization with thelive virus vaccine HSV-2ΔgE(gfp) (labeled as 1×imm) In contrast, higherantibody titers were detected after the second immunization (labeled as2×imm) that persisted for 3 and 5 months (FIG. 46).

Example 27 Neutralizing Antibody Response after One or Two Immunizationswith HSV-2ΔgE(gfp)

Female BALB/c mice at 5-6 weeks age were immunized IM in the calf musclewith HSV-2ΔgE(gfp) at 5×10³, 5×10⁴, or 5×10⁵ PFU Immunizations weregiven once or twice separated by 4 weeks. Serum was collected 4 weeksafter the first or 4 weeks after the second immunization and tested forneutralizing antibodies by incubating serial dilutions of serum with 200PFU of HSV-2 strain 2.12 for 1 h at 37° C. and measuring the virus titerby plaque assay on Vero cells. The end point titer was that dilution ofserum that reduced the virus titer by ≧50%. Legend: I immu, immunizedonce; II immu, immunized twice. n=5-10 animals per group.

Results

At each immunization dose, neutralizing antibody titers were higherafter the second immunization than the first. Titers were highest inmice immunized with 5×10⁵ PFU (FIG. 47).

Example 28 Protection of Mice by HSV-2ΔgE(gfp) is Dose Dependent

Mice were immunized with 5×10³, 5×10⁴, or 5×10⁵ PFU of HSV-2ΔgE(gfp)(labeled as gE-2null) given twice in the calf muscle separated by 4weeks. Four weeks after the second immunization, mice were challengedintravaginally with 5×10⁴ PFU of HSV-2 strain MS (approximately 10,000LD₅₀). Animals were followed for survival and were scored for vaginaldisease on a scale of 0-4, where 0 is no disease, and one point wasassigned for each of the following: erythma/swelling, exudate, hair lossin the perineal area, and ulcers or necrosis in the vaginal area(maximum score of 4 per animal per day). Moreover, animals wereevaluated for vaginal titers, which were determined by swabbing thevagina and titering virus by plaque assay on Vero cells Animals werealso evaluated for viral titers or viral DNA in dorsal root ganglia(DRG) 4 days post-infection or 35 days post-infection (labeled as latentviral load). DRG were harvested at either 4 days or 35 dayspost-challenge. For viral titers, the DRG samples were minced with smallscissors and pulverized using a pestle and half the sample was titeredon Vero cells. The remainder of the DRG sample was evaluated byreal-time quantitative PCR (RT qPCR) by amplifying the HSV-2 U_(s)9 DNA.Mouse adipsin DNA DNA was amplified in each well as a DNA control. PCRwas performed in 96-well qPCR plates using 2×FAST Taqman master mix(Applied Biosystems). Standard curves were prepared using purified HSV-2DNA (Advanced Biotechnologies) and mouse lung genomic DNA as a source ofthe adipsin gene (BioChain Institute). The standard curves were run intriplicate wells at 5, 50, 500, 5,000 and 50,000 copies of DNA. DRG DNA(Qiagen DNeasy) samples were run in duplicate and results are reportedas the number of HSV-2 DNA copies per 10⁵ adipsin genes. Primers foradipsin were: forward 5′-GCAGTCGAAGGTGTGGTTACG-3′-(SEQ ID NO: 20), andreverse 5′-GGTATAGACGCCCGGCTTTT-3′(SEQ ID NO: 21). Reporter dye andprobe for adipsin were: 5′-VIC-CTGTGGCAATGGC-3′MGBNFQ (SEQ ID NO: 22)(minor grove binder non-fluorescent quencher). Primers for HSV-2 Us9were: forward 5′-GCAGAAGCCTACTACTCGGAAA-3′ (SEQ ID NO: 23), reverse5′-CCATGCGCACGAGGAAGT-3′ (SEQ ID NO: 24). Reporter dye and probe for Us9were: 5′-6FAM-CGAGGCCAAC-3′-MGBNFQ (SEQ ID NO: 25). Primers for gpGAPDH(for studies described in FIG. 50 in guinea pigs) were: forward5′-CATGACAACTTCGGCATTGTG-3′ (SEQ ID NO: 26), reverse5′-TCTTCTGGGTGGCAGTGATG-3′ (SEQ ID NO: 27). Primers for HSV-2 gE were:forward 5′-CGTCTGGATGCGGTTTGAC-3′(SEQ ID NO: 28), and reverse5′-CTGGAAGCTGCGGGTGATAC-3′ (SEQ ID NO: 29). Reporter dye and probe forgE were: 6FAM-5′-ATGCGGATCTACGAAGC-3′-MGBNFQ (SEQ ID NO: 30). Primersfor GFP were: forward 5′-AGCAAAGACCCCAACGAGAA-3′ (SEQ ID NO: 31), andreverse 5′-GGCGGCGGTCACGAA-3′ (SEQ ID NO: 32). Reporter dye and probefor GFP were: 6FAM-5′-ATCACATGGTCCTGCTGG-3′-MGBNFQ (SEQ ID NO: 33).Reactions were performed using 5 μl of sample DNA in 25 μl volume usingthe TaqMan Gene Expression Master Mix (Applied Biosystems) and the ABI7500 Fast machine.

Results

No mock immunized mouse survived, while survival was 60% in miceimmunized with 5×10³ PFU, and 100% in mice immunized with 5×10⁴ or 5×10⁵PFU of the vaccine strain (labeled as gE-2null) (FIG. 48A). Vaginaldisease scores were highest in mock immunized mice, and declinedproportionally in mice immunized with 5×10³, 5×10⁴, or 5×10⁵ PFU of thevaccine strain. The number of animals surviving until days 6, 7 and 8 inthe mock immunized group is noted on the graph (FIG. 48B). Vaginaltiters were highest in mock immunized mice, and declined proportionallyin mice immunized with 5×10³, 5×10⁴, and 5×10⁵ PFU of the vaccinestrain.

D. Virus titers or viral load in the DRG measured by real-time qPCR. Thegraph on the left shows viral titers 4 days post-challenge in mockimmunized mice or HSV-2ΔgE(gfp) immunized mice (labeled as gE2-del).Titers were ˜4 log₁₀ in mock immunized compared with ˜1 log₁₀ inHSV-2ΔgE(gfp) immunized mice (P<0.001). The middle graph shows viralload in DRG at day 4. Mock immunized mice had between 6-7 log₁₀ copiesof HSV-2 DNA in DRG compared with ˜3 log₁₀ copies in HSV-2ΔgE(gfp)immunized mice (P<0.001). The right graph evaluates DRG 35 dayspost-challenge. No mock immunized mouse survived to day 35. The DRG ofHSV-2ΔgE(gfp) immunized mice (labeled as gE2-del) showed 2-3 log₁₀copies of HSV-2 DNA.

From the experiments shown in FIG. 48 it is concluded that theprotection of mice by HSV-2ΔgE(gfp) is dose dependent, with greaterprotection at 5×10⁵ PFU than at the lower immunizing doses, and that thevaccine strain provides substantial protection to the DRG against highdose challenge with HSV-2 strain MS.

Example 29 Assessment of DRG for Wild-Type or Vaccine Strain DNA

The day 35 DRG described in FIG. 48 were assessed for wild-type orvaccine strain DNA by amplifying the HSV-2 gE gene (present in wild-typevirus, but not the vaccine strain) or GFP (present in the vaccinestrain, but not wild-type virus (FIG. 49). The primers and probes aredescribed in Example 28.

Results

Wild-type DNA (gE) was detected in all 9 mice, while GFP DNA (vaccinestrain) was detected in one mouse only. Therefore, the DNA detected inthe right graph of FIG. 48D is predominantly from the challenge strainHSV-2 MS and not the vaccine strain.

Example 30 Intramuscular Immunization with HSV-2ΔgE(gfp) Given TwiceProvides Protection Against Death, Vaginal Disease and RecurrentInfection

Immunization studies were performed in female Hartley strain guinea pigs(175 to 225 grams at the time of first immunization) that were injectedin the calf muscle of the left hind leg two times at 4-week intervalswith either HSV-2ΔgE(gfp) (labeled as gE2-del) at 5×10⁵ PFU or with Verocell lysate. As a control for some experiments, 5 ug gC-2 subunitimmunogen was injected intramuscularly three times at 2-week intervalswith CpG and alum as adjuvants. Adjuvants for guinea pigs were CpG: TCGTCG TTG TCG TTT TGT CGT T (SEQ ID NO: 34) 100 μg per guinea pig wasmixed with alum 20 μg/ng protein in a 50 μl volume. For challengestudies, guinea pigs were infected intravaginally with HSV-2 MS strainat 5×10³ or 5×10⁵ PFU in 50 ul using a soft catheter to inject thevirus. Vaginal titers were measured 1 to 7 days post-infection byinserting a moistened swab into the vagina, and then placing the swab in1 ml of DMEM-10% FBS. Samples were stored at −70° C. until titers weredetermined by plaque assay Animals were observed daily, and diseaseseverity was scored as follows: 1 point for erythema, 2 points fordiscrete lesions, 3 points for coalesced lesions and 4 for ulcerativelesions. Disease that occurred 1 to 14 days post-infection wasconsidered as acute infection, while disease that developed 15 to 60days post-infection was considered as recurrent infection. At day 60,sacral DRG were harvested to evaluate for viral DNA copy number byreal-time qPCR, which was performed as described in FIG. 48 (Example28). Blood was collected from the lateral saphenous vein of the hindlimb at various times for antibody assays.

Results

All mock immunized guinea pigs challenged with 5×10³ or 5×10⁵ PFU died,while all HSV-2ΔgE(gfp) or gC-2 immunized guinea pigs survived (FIG.50A). Vaginal disease scores were highest in mock immunized animalschallenged with 5×10⁵ PFU, and lowest in animals immunized withHSV-2ΔgE(gfp) (FIG. 50B). Vaginal titers were highest in the mockimmunized animals and lowest in animals immunized with HSV-2ΔgE(gfp) orgC-2. No significant differences were detected comparing HSV-2ΔgE(gfp)with gC-2; however, both these groups were significantly different frommock immunized animals (P<0.001) (FIG. 50C). The table shows the numberof recurrences and the number of animals having a recurrence betweendays 15-49 post-infection. No mock immunized animal survived long enoughto assess recurrences, even at the lower challenge dose of 5×10³ PFU ofHSV-2 MS. Three of the 5 animals immunized with gC-2 had recurrences,and these 3 animals had a total of 10 recurrences that lasted a total of12 days. In contrast, only 1 of 10 animals immunized with HSV-2ΔgE(gfp)had a recurrent infection (P<0.001), which lasted for 2 days (FIG. 50D).DRG were harvested at the end of the experiment and assessed for HSV-2DNA by real-time qPCR. HSV-2 DNA at low levels was detected in 2 of 5guinea pigs immunized with gC-2, compared with 0 of 5 guinea pigsimmunized with HSV-2ΔgE(gfp) (FIG. 50E).

It is concluded that immunization with HSV-2ΔgE(gfp) given twice IM inthe calf separated by 4 weeks at a dose of 5×10⁵ PFU provides protectionagainst death, vaginal disease and recurrent infection.

Example 31 Two Immunizations With HSV-2ΔgE(gfp) After Recovery from aPrimary HSV-2 Genital Infection Produce Higher Titers to gC-2 and gD-2than One Immunization and is Effective in Reducing the Frequency orRecurrent Lessions

The therapeutic potential of the live virus vaccine was examined infemale Hartley strain guinea pigs (175 to 225 grams). 30 guinea pigswere infected intravaginally with HSV-2 MS strain at 10⁴ PFU in 50 ulusing a soft catheter. 17 animals survived the infection. Antibodytiters to gC-2 and gD-2 were determined 28 days post-infection in these17 animals to confirm infection. Only 11 animals had antibodies toeither gC-2 or gD-2, indicating infection in these 11 guinea pigs. 50days after the initial infection, the 11 guinea pigs were immunized IMin the calf muscle of the left hind leg twice separated by 28 days. 6animals were randomly assigned to receive two immunizations withHSV-2ΔgE(gfp) at 5×10⁵ PFU, while 5 animals were mock immunized twicewith Vero cell lysates. Animals were bled for antibodies 4 weeks aftereach immunization and antibodies measured to gC-2 (A) or gD-2 (B)Animals were scored for recurrent infections from day 16 after the firstimmunization until day 58 after the first immunization. One point wasassigned for each lesion observed (C). DRG were harvested for HSV-2 DNAby real-time qPCR at the end of the experiment.

Results

After recovery from acute infection, only 1 of 6 guinea pigs hadantibodies to gC-2 after the first immunization, compared to 5 of 6after the second immunization, indicating that two immunizations inducedhigher ELISA titers to gC-2 than one immunization (FIG. 51A). Afterrecovery from acute infection, 4 of 6 guinea pigs had gD-2 antibodiesafter both the first immunization and the second immunization; however,antibody titers were higher after the second immunization (P<0.01) (FIG.51B). Recurrent lesions developed significantly more often in mockimmunized than HSV-2ΔgE(gfp) immunized guinea pigs, indicating that thelive virus vaccine is effective in reducing the frequency of recurrentinfection (P<0.0001) (FIG. 51C). 3 of 5 DRG from mock immunized animalswere positive for HSV-2 DNA at the end of the experiment, compared with1 of 5 from HSV-2ΔgE(gfp) immunized animals (FIG. 51D).

It is concluded that when HSV-2ΔgE(gfp) is used as a therapeuticvaccine, two immunizations produce higher titers to gC-2 and gD-2 thanone immunization, and that the vaccine is effective in reducing thefrequency of recurrent lesions.

Having described the embodiments of the invention with reference to theaccompanying drawings, it is to be understood that the invention is notlimited to the precise embodiments, and that various changes andmodifications may be effected therein by those skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

What is claimed is:
 1. A method of inducing an anti-Herpes Simplex Virus(HSV) immune response in a subject comprising the steps of: (a)administering a therapeutically effective amount of a compositioncomprising an attenuated HSV strain to said subject, wherein saidattenuated HSV strain comprises a mutated HSV glycoprotein E, and (b)performing a second administration of said composition at saidtherapeutically effective amount comprising said attenuated HSV strainfollowing step (a), wherein the nucleic acid encoding the mutated HSV gEcomprises a single inactivating mutation resulting in the expression ofonly the first 123 amino acids of gE, wherein the first 123 amino acidsof gE correspond to amino acid positions 1-123 of SEQ ID NO: 2, andwherein said anti-HSV immune response induced is a protective and/ortherapeutic immune response.
 2. The method of claim 1, wherein saidattenuated HSV strain is an HSV-1 or HSV-2 strain.
 3. The method ofclaim 1, wherein said attenuated HSV strain a) has a defect in neuronalspread in the anterograde and retrograde directions; b) is deficient incell-to-cell spread; c) is replication-competent; or d) a combinationthereof.
 4. The method of claim 1, wherein said induced immune responseis an anti-HSV neutralizing antibody response.
 5. The method of claim 1,wherein said subject is infected by or is at risk for infection by HSV.6. The method of claim 1, wherein said initial administration is priorto exposure to HSV.
 7. The method of claim 1, wherein said initialadministration is after exposure to HSV.
 8. The method of claim 1,wherein said composition is administered intramuscularly, epidermally,subcutaneously, intravaginally, or via intra-respiratory mucosalinjection.
 9. A method of treating a Herpes Simplex Virus (HSV)infection in a subject comprising the steps of: (a) administering atherapeutically effective amount of a composition comprising anattenuated HSV strain to said subject, wherein said attenuated HSVstrain comprises a mutated HSV glycoprotein E, and (b) performing asecond administration of said composition at said therapeuticallyeffective amount comprising said attenuated HSV strain following step(a), wherein the nucleic acid encoding the mutated HSV gE comprises asingle inactivating mutation resulting in the expression of only thefirst 123 amino acids of gE, wherein the first 123 amino acids of gEcorrespond to amino acid positions 1-123 of SEQ ID NO:
 2. 10. The methodof claim 8, wherein said attenuated HSV strain is an HSV-1 or HSV-2strain.
 11. The method of claim 8, wherein said attenuated HSV strain a)has a defect in neuronal spread in the anterograde and retrogradedirections; b) is deficient in cell-to-cell spread; c) isreplication-competent; or d) a combination thereof.
 12. The method ofclaim 8, wherein said HSV infection is an HSV-1 infection.
 13. Themethod of claim 8, wherein said HSV infection is an HSV-2 infection. 14.The method of claim 8, wherein said composition is first administeredprior to exposure to HSV.
 15. The method of claim 8, wherein saidcomposition is first administered after exposure to HSV.
 16. The methodof claim 8, wherein said HSV infection is a primary HSV infection. 17.The method of claim 8, wherein said HSV infection is HSV encephalitis,an HSV neonatal infection, or HSV labialis.
 18. The method of claim 8,wherein said HSV infection is a genital, oral, or ocular HSV infection.19. The method of claim 8, wherein said HSV infection is a flare,recurrence, or HSV labialis following a primary HSV infection.
 20. Themethod of claim 8, wherein said composition is administeredintramuscularly, epidermally, subcutaneously, intravaginally, or viaintra-respiratory mucosal injection.
 21. A method of suppressing arecurrent Herpes Simplex Virus (HSV) infection in a subject comprisingthe steps of: (a) administering a therapeutically effective amount of acomposition comprising an attenuated HSV strain to said subject, whereinsaid attenuated HSV strain comprises a mutated HSV glycoprotein E, and(b) performing a second administration of said composition at saidtherapeutically effective amount comprising said attenuated HSV strainfollowing step (a), wherein the nucleic acid encoding the mutated HSV gEcomprises a single inactivating mutation resulting in the expression ofonly the first 123 amino acids of gE, wherein the first 123 amino acidsof gE correspond to amino acid positions 1-123 of SEQ ID NO:
 2. 22. Themethod of claim 21, wherein said attenuated HSV strain is an HSV-1 orHSV-2 strain.
 23. The method of claim 21, wherein said attenuated HSVstrain a) has a defect in neuronal spread in the anterograde andretrograde directions; b) is deficient in cell-to-cell spread; c) isreplication-competent; or d) a combination thereof.
 24. The method ofclaim 21, wherein said HSV infection is an HSV-1 infection.
 25. Themethod of claim 21, wherein said HSV infection is an HSV-2 infection.26. The method of claim 21, wherein said HSV infection is a genital,oral, or ocular HSV infection.
 27. The method of claim 21, wherein saidrecurrent HSV infection is HSV labialis following a primary HSVinfection.
 28. The method of claim 21, wherein said composition isadministered intramuscularly, epidermally, subcutaneously,intravaginally, or via intra-respiratory mucosal injection.
 29. Themethod of claim 1, wherein said immune response is a protective immuneresponse.
 30. The method of claim 1, wherein said immune response is atherapeutic immune response.
 31. The method of claim 9, wherein saidtreating said HSV infection comprises reducing the frequency ofrecurrent lesions.
 32. The method of claim 9, wherein said treating saidHSV infection comprises inducing rapid clearance of said HSV infectionin said subject.
 33. The method of claim 9, wherein said treating saidHSV infection comprises reducing the severity of said HSV infection insaid subject.
 34. The method of claim 21, wherein said suppressing arecurrent HSV infection comprises preventing latent infection of HSV insaid subject.